TW201833323A - Recombinant listeria vaccine strains and methods of using the same in cancer immunotherapy - Google Patents

Abstract

Provided herein are recombinant fusion polypeptides comprising an HPV16 antigenic peptide and an HPV18 antigenic peptide, wherein the HPV16 antigenic peptide and the HPV18 antigenic peptide are operably linked in tandem (e.g., fused to a PEST-containing peptide). Also provided are nucleic acids encoding such fusion polypeptides, recombinant bacteria or Listeria strains comprising such fusion polypeptides or such nucleic acids, and cell banks comprising such recombinant bacteria or Listeria strains. Also provided herein are methods of generating such fusion polypeptides, such nucleic acids, and such recombinant bacteria or Listeria strains. Also provided are immunogenic compositions, pharmaceutical compositions, and vaccines comprising such fusion polypeptides, such nucleic acids, or such recombinant bacteria or Listeria strains. Also provided are methods of inducing an anti-tumor-associated-antigen immune response in a subject, methods of inducing an anti-tumor or anti-cancer immune response in a subject, methods of treating a tumor or cancer in a subject, methods of preventing a tumor or cancer in a subject, and methods of protecting a subject against a tumor or cancer using such recombinant fusion polypeptides, nucleic acids, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines.

Description

Recombinant Listeria vaccine strain and method for using the same in cancer immunotherapy

The present invention is a recombinant Listeria vaccine strain and a method of using the same in cancer immunotherapy.

[0001] According to the CDC, based on data from 2008 to 2012, there are approximately 38,793 human papillomavirus (HPV)-related cancers occurring each year in the United States: approximately 23,000 in women and approximately 15,793 in men. HPV-related cancers include cervical cancer, anal cancer, head and neck cancer, or oropharyngeal cancer. [0002] Cervical cancer is the most common HPV-related cancer among women, while oropharyngeal cancer (the posterior of the throat, including the tongue and tonsils) is the most common HPV-related cancer among men. [0003] The E6 and E7 proteins from high-risk HPV 16 and HPV 18 are tumor proteins that act by stimulating the disruption of proteins that are critically regulated by many host cells. For example, HPV16 E6 associates with the host E6-AP ubiquitin-protein ligase and inactivates it by targeting the tumor suppressor TP53 and TP73 to the 26S proteasome for degradation, and this inactivation Increase DNA damage and chromosomal instability, and lead to cell proliferation and cancer development. [0004] Standard chemoradiation protocols commonly used in advanced cancer may be accompanied by significant toxicity. There is a need for improved therapeutic modalities in patients with HPV-associated cancers, and immunotherapy has the potential to reduce toxicity via a chemoradiation protocol and potentially enhance long-term disease control.

Methods and compositions for cancer immunotherapy are provided. In one aspect, provided herein is a recombinant Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to an HPV 16 antigen peptide and an HPV 18 antigen peptide Wherein the HPV 16 antigen peptide and the HPV 18 antigen peptide are operably linked in series. The HPV16 antigen peptide is HPV16 E6 antigen peptide or HPV16 E7 antigen peptide, and the HPV18 antigen peptide is HPV18 E6 antigen peptide or HPV18 E7 antigen peptide, as the case may be. The HPV16 antigen peptide is an HPV16 E7 antigen peptide, and the HPV18 antigen peptide is an HPV18 E7 antigen peptide, as the case may be. Such fusion polypeptides and nucleic acids encoding such fusion polypeptides are also provided. [0006] In another aspect, provided herein is an immunogenic composition, pharmaceutical composition or vaccine comprising a recombinant Listeria strain comprising a first reading comprising a fusion polypeptide encoding The nucleic acid of the frame, wherein the fusion polypeptide comprises a PEST-containing peptide fused to an HPV16 antigen peptide and an HPV18 antigen peptide, wherein the HPV 16 antigen peptide and the HPV 18 antigen peptide are operably linked in series. The HPV16 antigen peptide is HPV16 E6 antigen peptide or HPV16 E7 antigen peptide, and the HPV18 antigen peptide is HPV18 E6 antigen peptide or HPV18 E7 antigen peptide, as the case may be. The HPV16 antigen peptide is an HPV16 E7 antigen peptide, and the HPV18 antigen peptide is an HPV18 E7 antigen peptide, as the case may be. An immunogenic composition, pharmaceutical composition or vaccine comprising the fusion polypeptide or a nucleic acid encoding the fusion polypeptide is also provided. [0007] In another aspect, provided herein is a method of inducing an immune response against a tumor or cancer in an individual comprising administering to the individual a recombinant Listeria strain, the recombinant Listeria strain comprising the inclusion A nucleic acid encoding a first reading frame of a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to an HPV 16 antigen peptide and an HPV 18 antigen peptide, wherein the HPV 16 antigen peptide and the HPV 18 antigen peptide are operably linked in series. The HPV16 antigen peptide is HPV16 E6 antigen peptide or HPV16 E7 antigen peptide, and the HPV18 antigen peptide is HPV18 E6 antigen peptide or HPV18 E7 antigen peptide, as the case may be. The HPV16 antigen peptide is an HPV16 E7 antigen peptide, and the HPV18 antigen peptide is an HPV18 E7 antigen peptide, as the case may be. Also provided is a method of inducing an immune response against a tumor or cancer in an individual comprising administering to the individual an immunogenic composition, pharmaceutical composition or vaccine comprising such a recombinant Listeria strain. Also provided is a method of inducing an immune response against a tumor or cancer in an individual comprising administering to the individual the fusion polypeptide or a nucleic acid encoding the fusion polypeptide, the immunogenicity comprising the fusion polypeptide or the nucleic acid encoding the fusion polypeptide A composition, a pharmaceutical composition comprising the fusion polypeptide or the nucleic acid encoding the fusion polypeptide, or a vaccine comprising the fusion polypeptide or the nucleic acid encoding the fusion polypeptide. [0008] In another aspect, provided herein is a method of preventing or treating a tumor or cancer in an individual comprising administering to the individual a recombinant Listeria strain comprising a coding fusion The nucleic acid of the first open reading frame of the polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to the HPV16 antigen peptide and the HPV18 antigen peptide, wherein the HPV16 antigen peptide and the HPV18 antigen peptide are operably linked in series. The HPV16 antigen peptide is HPV16 E6 antigen peptide or HPV16 E7 antigen peptide, and the HPV18 antigen peptide is HPV18 E6 antigen peptide or HPV18 E7 antigen peptide, as the case may be. The HPV16 antigen peptide is an HPV16 E7 antigen peptide, and the HPV18 antigen peptide is an HPV18 E7 antigen peptide, as the case may be. Also provided is a method of preventing or treating a tumor or cancer in an individual comprising administering to the individual an immunogenic composition, pharmaceutical composition or vaccine comprising such a recombinant Listeria strain. Also provided is a method of preventing or treating a tumor or cancer in an individual comprising administering to the individual the fusion polypeptide, a nucleic acid encoding the fusion polypeptide, an immunogenic composition comprising the fusion polypeptide or the nucleic acid encoding the fusion polypeptide A pharmaceutical composition comprising the fusion polypeptide or the nucleic acid encoding the fusion polypeptide or a vaccine comprising the fusion polypeptide or the nucleic acid encoding the fusion polypeptide. [0009] In another aspect, provided herein is a cell bank comprising one or more recombinant Listeria strains, the recombinant Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein The fusion polypeptide comprises a PEST-containing peptide fused to an HPV16 antigen peptide and an HPV18 antigen peptide, wherein the HPV 16 antigen peptide and the HPV 18 antigen peptide are operably linked in series. The HPV16 antigen peptide is HPV16 E6 antigen peptide or HPV16 E7 antigen peptide, and the HPV18 antigen peptide is HPV18 E6 antigen peptide or HPV18 E7 antigen peptide, as the case may be. The HPV16 antigen peptide is an HPV16 E7 antigen peptide, and the HPV18 antigen peptide is an HPV18 E7 antigen peptide, as the case may be.

DEFINITIONS [0015] The terms "protein", "polypeptide" and "peptide" as used interchangeably herein are meant to mean polymeric acids of any length, including both coding and non-coding amino acids, and chemically or Amino acid modified or derivatized in a biochemical manner. The term includes polymers that have been modified, such as polypeptides having a modified peptide backbone. [0016] The protein is said to have "N-terminal" and "C-terminal". The term "N-terminus" relates to the origin of a protein or polypeptide capped with an amino acid having a free amine group (-NH2). The term "C-terminus" relates to the end of an amino acid chain (protein or polypeptide) terminated by a free carboxyl group (-COOH). [0017] The term "fusion protein" refers to a protein comprising two or more than two peptides joined together by peptide bonds or other chemical bonds. Peptides can be directly linked together by peptide bonds or other chemical bonds. For example, a chimeric molecule can be recombinantly expressed in the form of a single-stranded fusion protein. Alternatively, the peptides may be joined together by a "linker", such as one or more amino acids between two or more than two peptides or another suitable linker. [0018] The terms "nucleic acid" and "polynucleotide" as used interchangeably herein are meant to refer to nucleotides of polymeric form of any length, including ribonucleotides, deoxyribonucleotides or the like or Modified form. It includes single, double or multiple strands of DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids and contains purine bases, pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural or derived a polymer of nucleotide bases. [0019] The nucleic acid is said to have a "5' end" and a "3' end" because the single nucleotide is reacted to produce an oligonucleotide such that a single nucleotide pentose ring is 5' phosphate. The ester is attached in one direction via a phosphodiester bond to the 3' oxygen of its adjacent mononucleotide pentose ring. If the 5' phosphate of the oligonucleotide is not linked to the 3' oxygen of the mononucleotide pentose ring, the end of the oligonucleotide is referred to as the "5' end". If the 3' oxygen of the oligonucleotide is not linked to the 5' phosphate of another mononucleotide pentose ring, the end of the oligonucleotide is referred to as the "3' end". Even within the larger oligonucleotide, the nucleic acid sequence can be said to have a 5' end and a 3' end. In a linear or circular DNA molecule, a discontinuous element is said to be "upstream" or 5' of the "downstream" or 3' element. [0020] "Codon optimization" refers to a method of modifying a nucleic acid sequence to confer enhanced expression in a particular host cell by replacing the native with the more frequently or most frequently used codons in the gene of the host cell. At least one codon of the sequence is carried out while maintaining the native amino acid sequence. For example, a polynucleotide encoding a fusion polypeptide can be modified to replace a codon that is more frequently used in a given Listeria cell or any other host cell than in a naturally occurring nucleic acid sequence. The codon usage table is readily available, for example, in the "Cryptography Database". The best codon used by Listeria monocytogenes for each amino acid is shown in US 2007/0207170, which is hereby incorporated by reference in its entirety for all purposes. These tables can be modified in a number of ways. See Nakamura et al. (2000)Nucleic Acids Research 28:292, which is hereby incorporated by reference in its entirety for all purposes. Computer algorithms for codon-optimizing specific sequences for expression in a particular host are also available (see, for example, Gene Forge). [0021] The term "plastid" or "vector" includes any known delivery vehicle, including bacterial delivery vehicles, viral vector delivery vehicles, peptide immunotherapy delivery vehicles, DNA immunotherapy delivery vehicles, episomal plastids, integrative profiles. Body or phage vector. The term "vector" refers to a construct that is capable of delivering in a host cell and, where appropriate, one or more fusion polypeptides. [0022] The term "additional plastid" or "exochromosomal" refers to a physical separation from chromosomal DNA (ie, episomal or extrachromosomal and not integrated into the host cell genome) and independent of chromosomal DNA. A replicating nucleic acid vector. The plastids can be linear or circular and can be single or double stranded. Additional plastids may be retained in multiple copies of the host cell cytoplasm (eg, Listeria), thereby causing any gene of interest to be amplified in the episomal body. [0023] The term "genomic integration" refers to the introduction of a nucleic acid into a cell such that the nucleotide sequence is integrated into the genome of the cell and can be inherited by its progeny. Any protocol can be used to stably incorporate nucleic acids into the genome of a cell. [0024] The term "stable maintenance" refers to the maintenance of a nucleic acid molecule or plastid in the absence of selection (eg, antibiotic selection) for at least 10 generations without detectable loss. For example, the time period can be at least 15, 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 150, at least 200, At least 300 generations or at least 500 generations. Stable maintenance can refer to nucleic acid molecules or plastids in cellsIn vitro (for example, in culture) maintain stability, maintain stability in vivo, or both. [0025] An "open reading frame" or "ORF" is a portion of DNA containing a base sequence that can potentially encode a protein. As an example, the ORF can be located between the start coding sequence of the gene (start codon) and the stop codon sequence (stop codon). A "promoter" is a regulatory region of DNA that typically comprises a TATA(R) capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site of a particular polynucleotide sequence. The promoter may additionally contain additional regions that affect the rate of transcription initiation. The promoter sequences disclosed herein modulate the transcription of operably linked polynucleotides. The promoter may be in one or more of the cell types disclosed herein (eg, eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, single cell stage embryos, differentiated cells, or a combination thereof) Active. The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a time-restricted promoter (eg, a developmentally regulated promoter), or a sterically restricted promoter (eg, cell-specific or tissue-specific) Promoter). Examples of promoters can be found, for example, in WO 2013/176772, which is incorporated herein by reference in its entirety. [0027] "Operably linked" or "operably connected" means that two or more components (eg, a promoter and another sequence element) are joined in such a way that both components function properly and allow such At least one of the components may be capable of mediating functions applied to at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulators. An operably linked sequence can include such sequences that are adjacent to each other or such sequences that are in trans-acting (eg, regulatory sequences can function at intervals to control transcription of the coding sequence). [0028] A "sequence identity" or "consistency" reference in the context of two polynucleotide or polypeptide sequences, the same residues in the two sequences when the alignment is maximized within the specified comparison window . When reference proteins are used to use percent sequence identity, it is often confirmed that the inconsistent residue positions differ in conservative amino acid substitutions in which the amino acid residues are substituted with other chemical properties (such as charge or hydrophobicity). Amino acid residues and therefore do not alter the functional properties of the molecule. When the sequence differs by conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. The difference is that such conservatively substituted sequences are said to have "sequence similarity" or "similarity." The manner in which this adjustment is made is well known to those skilled in the art. This typically involves scoring conservative substitutions as partial but not all mismatches, thereby increasing the percent sequence identity. Thus, for example, when a score of 1 is given for a consensus amino acid and a score of 0 is given for a non-conservative substitution, a score between 0 and 1 is given for a conservative substitution. The score for conservative substitution is calculated, for example, as implemented in the program PC/GENE (Intelligenetics, Mountain View, California). [0029] "Sequence Consistency Percent" refers to a value (the maximum number of perfectly matched residues) determined by comparing two optimal alignment sequences within a comparison window, wherein the polynucleotide sequence portion of the comparison window Additions or deletions (i.e., gaps) may be included as compared to a reference sequence for optimal alignment of two sequences (which does not include additions or deletions). The percentage is calculated by measuring the number of positions of a consensus nucleic acid base or an amino acid residue in two sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and the result Multiply by 100 to get the percent sequence identity. Unless otherwise stated (eg, a shorter sequence includes a joined heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared. [0030] Unless otherwise stated, sequence identity/similarity values refer to values obtained using GAP Version 10 using the following parameters: % identity of nucleotide sequences and % similarity using gap weights of 50 and length weights of 3, and Nwsgapdna.cmp scoring matrix; % identity and similarity % of amino acid sequences use gap weight 8 and length weight 2, and BLOSUM62 scoring matrix; or any equivalent procedure thereof. "Equivalent procedure" includes producing an alignment with a uniform nucleotide or amino acid residue match for any two sequences in question and consistent with the sequence when compared to the corresponding alignment generated by GAP Version 10. Any sequence comparison procedure for a consistent percentage of sex. [0031] The term "conservative amino acid substitution" refers to the replacement of an amino acid typically present in a sequence with a different amino acid of similar size, charge or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine or leucine to another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and aspartame, or between glycine and Between the serines. In addition, substitution of a basic residue such as an amino acid, arginine or histidine for another, or substitution of an acidic residue such as aspartic acid or glutamic acid for another acidic residue is conservative Replace the additional examples. Examples of non-conservative substitutions include the substitution of non-polar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine or methionine to such as cysteine, bran A polar (hydrophilic) residue of an amine, glutamic acid or lysine and/or a polar residue substituted with a non-polar residue. A typical amino acid classification is outlined below.[0032] A "homologous" sequence (eg, a nucleic acid sequence) refers to a sequence that is identical or substantially similar to a known reference sequence such that it is at least 50%, at least 55%, at least 60%, at least with a known reference sequence. 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical. [0033] The term "wild type" refers to an entity having a structure and/or activity as seen in a normal (relative to a mutated, ill, altered or the like) state or situation. Wild-type genes and polypeptides are often present in a variety of different forms, such as dual genes. [0034] The term "isolated" with respect to proteins and nucleic acids means that the protein and nucleic acid are relatively purified with respect to other bacteria, viruses or cellular components normally present in situ, up to and including the protein and the polynucleotide. A substantially pure preparation. The term "isolated" also includes those that do not have a naturally occurring counterpart, have been chemically synthesized and are therefore substantially free of contamination by other proteins or nucleic acids, or have been associated with most of their other cellular components (eg, other cells) Proteins and nucleic acids isolated or purified by proteins, polynucleotides or cellular components). [0035] An "exogenous" or "heterologous" molecule or sequence is a molecule or sequence that is not normally expressed in a cell or that is not normally present in the cell in that form. It is usually present to include the specific developmental stages of the cell and the presence of environmental conditions. For example, an exogenous or heterologous molecule or sequence may comprise a mutated version of a corresponding endogenous sequence within a cell, or may comprise a sequence corresponding to an endogenous sequence within a cell but in a different form (ie, , not in the chromosome). An exogenous or heterologous molecule or sequence in a particular cell may also be a molecule or sequence derived from a species different from the reference species of the cell or from a different organism within the same species. For example, in the case of a Listeria strain exhibiting a heterologous polypeptide, the heterologous polypeptide may be a polypeptide that is not native or endogenous to the Listeria strain, and is typically not expressed by the Listeria strain. a polypeptide derived from a source other than the Listeria strain, derived from a polypeptide of a different organism within the same species. In contrast, an "endogenous" molecule or sequence or "native" molecule or sequence is a molecule or sequence that is normally present in a particular cell in that form at a particular developmental stage under particular environmental conditions. [0037] The term "variant" refers to an amino acid or nucleic acid sequence (or organism or tissue) (eg, a splice variant) that differs from most populations but is still sufficiently similar to one of the common modes considered to be one of them. [0038] The term "isotope" refers to a form of a molecule (eg, a protein) that differs only slightly from another isoform or pattern (eg, of the same protein). For example, a protein isoform can be produced by a different but related gene, which can be produced by the same gene by alternative splicing, or it can be produced by a single nucleotide polymorphism. [0039] When referring to a protein, the term "fragment" means a protein that is shorter than a full length protein or has fewer amino acids. When referring to a nucleic acid, the term "fragment" means a nucleic acid that is shorter or has fewer nucleotides than the full length nucleic acid. The fragment may be, for example, an N-terminal fragment (i.e., one portion of the C-terminus of the protein is removed), a C-terminal fragment (i.e., one portion of the N-terminus of the protein is removed) or an internal fragment. Fragments can also be, for example, functional fragments or immunogenic fragments. [0040] When referring to a protein, the term "analog" means a protein that differs from a naturally occurring protein by a difference in a conserved amino acid, a modification that does not affect the amino acid sequence, or both. [0041] The term "functional" refers to the innate ability of a protein or nucleic acid (or a fragment, isoform or variant thereof) to exhibit biological activity or function. Such biological activity or function can include, for example, the ability to elicit an immune response when administered to an individual. Such biological activity or function can also include, for example, binding to an interaction partner. In the case of functional fragments, isoforms or variants, such biological functions may actually change (eg, with respect to their specificity or selectivity), but retain substantial biological function. [0042] The term "immunogenicity" or "immunogenic" refers to the innate ability of a molecule (eg, a protein, nucleic acid, antigen, or organism) to elicit an immune response in an individual when administered to an individual. . Immunogenicity can be measured, for example, by the greater number of antibodies relative to the molecule, the greater diversity of antibodies relative to the molecule, the greater number of T cells specific for the molecule, the cytotoxicity to the molecule or The helper T cell response is larger and its similar metrics. [0043] The term "antigen" is used herein to mean when placed in contact with an individual or organism (eg, when present in an individual or organism or when detected by an individual or organism), causing an individual or A substance in an organism that detects an immune response. The antigen can be, for example, a lipid, a protein, a carbohydrate, a nucleic acid, or a combination and variations thereof. For example, "antigenic peptide" refers to a peptide that, when present in or detected by an individual or organism, causes an established immune response in the individual or organism. For example, such "antigenic peptides" may encompass proteins that are loaded onto and present on MHC class I and/or class II molecules on the surface of a host cell, and that are immune cells from the host Identification or detection, thereby creating an immune response against the protein. Such immune responses can also be extended to other cells in the host, such as ill cells (eg, tumors or cancer cells) that exhibit the same protein. The term "antigenic determinant" refers to a site on an antigen that is recognized by the immune system (eg, bound to an antibody). An epitope can be formed by an adjacent amino acid or a non-adjacent amino acid that is ligated by a tertiary folding of one or more proteins. An epitope (also known as a linear epitope) formed by an adjacent amino acid is typically retained upon exposure to a denaturing solvent, while an epitope determined by tertiary folding (also known as a conformational epitope) is typically Loss when treated with a denaturing solvent. An epitope typically comprises at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial configuration. Methods for determining the spatial configuration of an epitope include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris, (1996), which is incorporated herein by reference in its entirety for all purposes. [0045] The term "mutation" refers to any change in the structure of a gene or protein. For example, a mutation can result from deletion, insertion, substitution, or rearrangement of a chromosome or protein. "Insert" alters the number of nucleotides in a gene or the number of amino acids in a protein by adding one or more additional nucleotides or amino acids. "Deletion" alters the number of nucleotides in a gene or the number of amino acids in a protein by reducing one or more additional nucleotides or amino acids. [0046] A "frame shift" mutation in DNA occurs when a nucleotide is added or the reading frame of the altered gene is lost. The reading frame consists of a group of 3 bases each encoding an amino acid. The in-frame transfer mutation shifts the grouping of these bases and alters the encoding of the amino acid. The resulting protein is usually non-functional. Insertions and deletions can each be a transfer mutation in the reading frame. [0047] A "missing" mutation or substitution refers to a change in an amino acid of a protein or a point mutation in a single nucleotide that causes a change in the encoded amino acid. A point mutation in a single nucleotide that causes an amino acid change is a "non-synonymous" substitution in the DNA sequence. Non-synonymous substitutions can also cause "nonsense" mutations in which the codon is changed to an early stop codon that causes the resulting protein to be truncated. In contrast, a "synonymous" mutation in DNA is a mutation that does not alter the amino acid sequence of the protein (due to degeneracy of the code). [0048] The term "somatic mutation" includes genetic alterations obtained by cells other than germ cells (eg, sperm or eggs). Such mutations can be transmitted to the progeny of the mutant cell during cell division but are not heritable. In contrast, reproductive mutations occur in germ cell lines and can be passed on to the next generation of offspring. [0049] The term "in vitro" refers to an artificial environment and processes or reactions that occur within an artificial environment (eg, a test tube). [0050] The term "in vivo" refers to a natural environment (eg, a cell or organism or body) and processes or reactions that occur within the natural environment. [0051] The composition or method of "comprising" or "comprising" one or more of the recited elements may include other elements not specifically described. For example, a "contained" or "included" protein composition can contain the protein alone or in combination with other ingredients. [0052] A range of specified values includes all integers within the range or defining the range, as well as sub-ranges defined by integers within the range. [0053] Unless otherwise apparent from the context, the term "about" encompasses a value within the standard measurement error margin (eg, SEM) of the stated value or ± 0.5%, 1%, 5%, or 10% relative to the specified value. Change. The singular articles "a", "an", "the" For example, the term "an antigen" or "at least one antigen" can include a plurality of antigens, including mixtures thereof. [0055] Statistically significant means p ≤ 0.05. I. Overview [0056] Provided herein are recombinant fusion polypeptides comprising an HPV 16 antigen peptide and an HPV 18 antigen peptide, wherein the HPV 16 antigen peptide and the HPV 18 antigen peptide are operably linked in tandem (eg, fused to a PEST-containing peptide). The HPV16 antigen peptide is HPV16 E6 antigen peptide or HPV16 E7 antigen peptide, and the HPV18 antigen peptide is HPV18 E6 antigen peptide or HPV18 E7 antigen peptide, as the case may be. The HPV16 antigen peptide is an HPV16 E7 antigen peptide, and the HPV18 antigen peptide is an HPV18 E7 antigen peptide, as the case may be. Also provided herein are nucleic acids encoding such fusion polypeptides; recombinant bacteria or Listeria strains comprising such fusion polypeptides or such nucleic acids; cell banks comprising such recombinant bacteria or Listeria strains; Such immunogenic compositions, pharmaceutical compositions and vaccines of such recombinant nucleic acids or such recombinant bacteria or Listeria strains; and production of such fusion polypeptides, such nucleic acids and such recombinant bacteria or Listeria strains method. Also provided are methods for inducing an immune response against an anti-tumor antigen in an individual, methods for inducing an anti-tumor or anti-cancer immune response in an individual, methods for treating a tumor or cancer in an individual, methods for preventing tumors or cancer in an individual, and protection The subject is free of tumor or cancer by methods such recombinant recombinant fusion polypeptides, nucleic acids, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines. [0057] Most HPV-related cancers are attributable to type 16 and type 18 HPV. However, HPV-associated cancers are generally not positive for both type 16 HPV and type 18 HPV. Because of the rare HPV16-positive and HPV18-positive patients, there is a lack of motivation to produce immunotherapy that targets both HPV16-specific and HPV18-specific antigens. This is especially true for Listeria-based immunotherapeutic platforms, which may have limited capacity for the number and size of nucleic acid sequences encoding insertable antigenic peptides. However, the evidence provided in the Examples herein demonstrates that it is bioengineered to secrete Listeria-based immunotherapy from an HPV-type antigenic peptide (eg,Lm Techniques can unexpectedly increase overall survival by twelve months in patients with cancer or tumors associated with different types of HPV. [0058]Lm The technology has the following mechanism of action: it also has potent innate immune stimulation, delivering the target peptide directly to the cytosol of dendritic cells and antigen presenting cells, producing a target T cell response, and by regulation in the tumor microenvironment Sex T cells and bone marrow-derived suppressor cells reduce immunosuppression. Multiple treatments can be given and/or combined without neutralizing antibodies.Lm Technology can be used, for example, in vivo, attenuated, bioengineeredLm Bacteria stimulate the immune system to treat tumor cells as potentially bacterially infected cells and target them for clearance. The technical method can begin with a live attenuated Listeria strain, and can add, for example, multiple copies of a plastid encoding a fusion protein sequence comprising, for example, Listeria lysin O (LLO, conjugated to the antigen of interest). Listeriolysin O) A fragment of the molecule. The fusion protein is present inside the antigen presenting cellListeria secretion. This causes stimulation of the branch of the innate and adaptive immune system, which reduces the mechanism of tumor defense and makes the immune system easier to attack and destroy cancer cells. II. Recombinant Fusion Polypeptides [0059] Disclosed herein are recombinant fusion polypeptides comprising a PEST-containing peptide fused to an HPV 16 antigen peptide and an HPV 18 antigen peptide, wherein the HPV 16 antigen peptide and the HPV 18 antigen peptide are operably linked in series. Also disclosed herein are recombinant fusion polypeptides comprising an HPV 16 antigen peptide and an HPV 18 antigen peptide, wherein the HPV 16 antigen peptide and the HPV 18 antigen peptide are operably linked in tandem, and wherein the fusion polypeptide does not comprise a PEST-containing peptide. Also provided herein are recombinant fusion polypeptides comprising a bacterial secretion sequence, a ubiquitin (Ub) protein and two or more than two antigenic peptides from the N-terminus to the C-terminus (ie, in tandem, such as Ub-peptide 1 Peptide 2), wherein the HPV 16 antigen peptide and the HPV 18 antigen peptide, wherein the HPV 16 antigen peptide and the HPV 18 antigen peptide are operably linked in series. Alternatively, a combination of separate fusion polypeptides can be used in which each antigen peptide is fused to its own secretory sequence and Ub protein (eg, Ub1-peptide 1; Ub2-peptide 2). Nucleic acids encoding such recombinant fusion polypeptides (referred to as pocket gene constructs) are also disclosed. Such pocket gene nucleic acid constructs may further comprise two or more open reading frames by the Xiain-Dalgano ribosome binding site (Shine-Dalgarno ribosome) between the open reading frames. Binding site) Nucleic acid sequence linkage. For example, the pocket gene nucleic acid construct can further comprise two to four open reading frames joined by a Xiain-Dalgano ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different polypeptide. In some nucleic acid constructs, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis. [0063] The bacterial signal sequence can be a Listeria signal sequence, such as a Hly or ActA signal sequence, or any other known signal sequence. In other cases, the signal sequence can be a sequence of LLO signals. The signal sequence can be bacterial and can be native to the host bacterium (eg, Listeria monocytogenes)Listeria monocytogenes ), the secA1 signal peptide), or may be foreign to the host bacteria. Specific examples of signal peptides include Lactococcus lactis (Lactococcus lactis Usp45 signal peptide; from Bacillus anthracis (Bacillus anthracis a protective antigen signal peptide; a secA2 signal peptide from Listeria monocytogenes, such as a p60 signal peptide; and a Tat signal peptide, such as Bacillus subtilis (B. subtilis A Tat signal peptide (eg, PhoD). In a specific example, the secretion signal sequence is from a Listeria protein, such as ActA₃₀₀ Secretory signal or ActA₁₀₀ Secretory signal. [0064] Ubiquitin can be, for example, a full length protein. The ubiquitin expressed from the nucleic acid constructs provided herein can be cleaved by a hydrolase upon entry into the cytosol of the host cell cytosol and cleavage at the carboxy terminus by the remainder of the recombinant fusion polypeptide expressed from the nucleic acid construct. This releases the amino terminus of the fusion polypeptide to produce a peptide in the host cell cytosol. [0065] The selection, variation, and alignment of antigenic peptides within a fusion polypeptide are discussed in detail elsewhere herein, and HPV 16 and HPV 18 antigen peptides are discussed in more detail elsewhere herein. [0066] A recombinant fusion polypeptide can comprise one or more tags. For example, a recombinant fusion polypeptide can comprise one or more peptide tags at the N-terminus and/or C-terminus of a combination of HPV 16 antigen peptides and HPV 18 antigen peptides operably linked in tandem. The tag can be fused directly to the antigenic peptide or linked to the antigenic peptide via a linker (examples of which are disclosed elsewhere herein). Examples of tags include the following: FLAG tags, 3 x FLAG tags; His tags, 6 x His tags; and SIINFEKL tags. An exemplary SIINFEKL tag is set forth in SEQ ID NO: 16 (encoded by any of the nucleic acids set forth in SEQ ID NOs: 1-15). An exemplary 3xFLAG tag is set forth in SEQ ID NO: 32 (encoded by any of the nucleic acids set forth in SEQ ID NOs: 17-31). Other labels include chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin ( Thioredoxin; TRX) and poly(NANP). A particular recombinant fusion polypeptide comprises a C-terminal SIINFEKL tag. Such tags may allow for easy detection of recombinant fusion proteins, confirmation of secretion of recombinant fusion proteins, or for tracking the immunogenicity of secreted fusion polypeptides by tracking immune responses to such "tag" sequence peptides. Such immune responses can be monitored using a number of reagents, including, for example, monoclonal antibodies and DNA or RNA probes that are specific for such tags. The recombinant fusion polypeptides disclosed herein can be expressed by recombinant Listeria strains, or can be expressed and isolated from other vectors and cellular systems for protein expression and isolation. A recombinant Listeria strain comprising a peptide exhibiting such an antigen can be used, for example, in an immunogenic composition comprising such recombinant Listeria and in a vaccine comprising a recombinant Listeria strain and an adjuvant. In a host cell system of a Listeria strain and a host cell system other than Listeria, one or more antigenic peptides are expressed as a fusion polypeptide using a truncated form of a non-hemolytic LLO, ActA or PEST-like sequence. It causes an increase in the immunogenicity of the antigenic peptide. Nucleic acids encoding such recombinant fusion polypeptides are also disclosed. The nucleic acid can be in any form. The nucleic acid may comprise or consist of DNA or RNA and may be single or double stranded. Nucleic acids can be in the form of plastids, such as episomal, multi-replicative plastids or integrative plastids. Alternatively, the nucleic acid can be in the form of a viral vector, a phage vector, or in a bacterial artificial chromosome. Such nucleic acids can have an open reading frame or can have two or more open reading frames (eg, an open reading frame encoding a recombinant fusion polypeptide and a second open reading frame encoding a metabolic enzyme). In one example, such nucleic acids can comprise two or more than one open reading frames joined by a Xiain-Dalkano ribosome binding site nucleic acid sequence between each open reading frame. For example, a nucleic acid can comprise two to four open reading frames joined by a Xiain-Dalkano ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different polypeptide. In some nucleic acids, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis. A. Antigen Peptides [0069] Antigenic peptides as used herein may include any HPV16-specific peptide and any combination of HPV18-specific peptides. Such peptides can be HPV16 specific and HPV 18 specific full length proteins or fragments thereof. Exemplary HPV 16 specific and HPV 18 specific proteins include E6 and E7 proteins from HPV 16 and HPV 18 . E6 and E7 proteins are tumor proteins that act by stimulating the destruction of proteins that are critically regulated by many host cells. Examples of HPV16 and HPV18 E6 and E7 proteins include HPV16 E7 (GenBank accession numbers AHK23257 and AAD33253; 98 amino acid proteins), HPV16 E6 (GenBank accession numbers AHK23256 and AAD33252; 158 amino acid proteins), HPV18 E7 (GenBank accession numbers AGM34461 and P06788; 105 amino acid proteins) and HPV18 E6 (GenBank accession number P06463; 158 amino acid proteins). An exemplary HPV16 E7 protein is set forth in SEQ ID NO: 96 (encoded by the DNA sequence set forth in SEQ ID NO: 95) and the exemplary HPV18 E7 protein is set forth in SEQ ID NO: 98 (by SEQ ID NO: 97 The DNA sequence code described in the above). A suitable HPV16 E7 peptide can be, for example, a protein that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 96 or a fragment thereof. A suitable HPV18 E7 peptide can be, for example, a protein that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 98 or a fragment thereof. [0070] A fusion polypeptide can include at least two antigenic peptides. For example, a fusion polypeptide can include 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigenic peptides. Each antigenic peptide may have any length sufficient to induce an immune response, and each antigenic peptide may be of the same length or the antigenic peptides may have different lengths. For example, the antigenic peptides disclosed herein can be about 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100. , 100-110, 110-120, 120-130, 130-140, 140-150, 150-160 or 95-160 amino acids. [0071] Each antigenic peptide may also be hydrophilic, or may be scored at or below a certain hydrophilicity threshold, which predicts sequestability in Listeria monocytogenes or another bacterial of interest. For example, the antigenic peptide can be scored by the Kyte and Doolittle Hydrophilicity Index 21 amino acid window, and all scores above the cutoff (approximately 1.6) can be excluded because it is unlikely to be increased by mononuclear spheres. Listeria monocytogenes secretion. Likewise, the combination of antigenic peptides or fusion polypeptides can be hydrophilic or can be scored at or below a certain hydrophilicity threshold, which can be predicted to be secreted in Listeria monocytogenes or another bacterial of interest. Sex. The antigenic peptides can be linked together in any manner. For example, antigenic peptides can be fused directly to each other without an intervening sequence. Alternatively, the antigenic peptides may be indirectly linked to one another via one or more linkers, such as peptide linkers. In some cases, pairs of adjacent antigenic peptides can be directly fused to each other, and other antigenic peptides can be indirectly linked to one another via one or more linkers. The same linker can be used between pairs of adjacent antigenic peptides, or any number of different linkers can be used between different pairs of adjacent antigenic peptides. Alternatively, one linker can be used between a pair of adjacent antigenic peptides, or multiple linkers can be used between a pair of adjacent antigenic peptides. Any suitable sequence can be used for the peptide linker. As an example, the length of the linker sequence can be, for example, from 1 to about 50 amino acids. Some linkers can be hydrophilic. The linker can be used for different purposes. For example, linkers can be used to increase bacterial secretion, promote antigen processing, increase the flexibility of the fusion polypeptide, increase the rigidity of the fusion polypeptide, or be used for any other purpose. In some cases, different amino acid linker sequences are distributed between antigenic peptides, or different nucleic acids encoding the same amino acid linker sequence are distributed between antigenic peptides (eg, SEQ ID NOS: 84-94) such that Minimize duplication. This can also be used to reduce the secondary structure, thereby allowingLm Efficient transcription, translation, secretion, maintenance or stabilization of other suitable peptide linker sequences for a nucleic acid encoding a fusion polypeptide (eg, a plastid) within a population of recombinant vector strains can be selected, for example, based on one or more of the following factors: (1) it can adopt a flexible extended configuration; (2) it cannot adopt a secondary structure that can interact with a functional epitope on an antigen peptide; and (3) lacks a possible reaction with a functional epitope Hydrophobic or charged residue. For example, a peptide linker sequence can contain Gly, Asn, and Ser residues. Other near neutral amino acids such as Thr and Ala can also be used in the linker sequence. Amino acid sequences useful as linkers include the amino acid sequences disclosed below: Maratea et al. (1985)Gene 40:39-46; Murphy et al. (1986)Proc Natl Acad Sci USA 83:8258-8262; US 4,935,233; and US 4,751,180, each of which is incorporated herein by reference in its entirety for all purposes. Specific examples of linkers include the linkers in the table below (each of which can be used alone as a linker, in a linker comprising a sequence repeat, or for further inclusion of one or more other Sequence linkers), although other linkers are also foreseen (see, for example, Reddy Chichili et al. (2013)Protein Science 22: 153-167, which is hereby incorporated by reference in its entirety for all purposes. Unless specified, "n" indicates the number of undetermined repetitions in the listed linkers.B. PEST-Containing Peptides [0074] The recombinant fusion proteins disclosed herein comprise a PEST-containing peptide. The PEST-containing peptide may be at the amino terminus (N-terminus) of the fusion polypeptide (ie, at the N-terminus of the antigenic peptide) and may be at the carboxy terminus (C-terminus) of the fusion polypeptide (ie, at the C-terminus of the antigenic peptide), or It can be embedded in an antigen peptide. In some recombinant Listeria strains and methods, the PEST-containing peptide is not part of the fusion polypeptide and is separate from the fusion polypeptide. Fusions of antigenic peptides to PEST-like sequences, such as LLO peptides, enhance the immunogenicity of the antigenic peptide and increase cell-mediated and anti-tumor immune responses (i.e., increase cell-mediated and anti-tumor immunity). See, for example, Singh et al. (2005)J Immunol 175(6): 3663-3673, which is hereby incorporated by reference in its entirety for all purposes. PEST-containing peptides are peptides comprising a PEST sequence or a PEST-like sequence. The PEST sequence in eukaryotic proteins has long been identified. For example, it is generally, but not necessarily, always flanked by a cluster containing a number of positively charged amino acids rich in proline (P), glutamic acid (E), serine (S) and threonine ( The protein of the amino acid sequence (PEST) of T) has a rapid intracellular half-life (Rogers et al. (1986)Science 234: 364-369, which is incorporated herein by reference in its entirety for all purposes. In addition, these sequences have been reported to target proteins to the ubiquitin-proteasome pathway for degradation (Rechsteiner and Rogers (1996)Trends Biochem. Sci 21:267-271, which is incorporated herein by reference in its entirety for all purposes. This pathway is also used by eukaryotic cells to produce immunogenic peptides that bind to MHC class I, and it has been hypothesized that PEST sequences are abundant in eukaryotic proteins that produce immunogenic peptides (Realini et al. (1994)FEBS Lett. 348: 109-113, which is hereby incorporated by reference in its entirety for all purposes. Prokaryotic proteins usually do not contain a PEST sequence because they do not have this enzymatic pathway. However, PEST-like sequences rich in amino acid glutamic acid (P), glutamic acid (E), serine (S) and threonine (T) have been reported at the amine end of LLO and have been reported. It is reported to be essential for the pathogenicity of Listeria monocytogenes (Decatur and Portnoy (2000)Science 290: 992-995, the entire disclosure of which is hereby incorporated by reference in its entirety in its entirety. The presence of this PEST-like sequence in the LLO is targeted by the proteolytic mechanism of the host cell to target the protein for destruction, such that once the LLO has functioned and promotes the monocytogenes, Listeria monocytogenes or phagocytosis The body vacuole escapes and the LLO is destroyed before it can damage the cells. Identification of PEST and PEST-like sequences is well known in the art and is described, for example, in Rogers et al. (1986).Science 234 (4774): 364-378, and Rechsteiner and Rogers (1996)Trends Biochem. Sci 21:267-271, each of which is incorporated herein by reference in its entirety for all purposes. PEST or PEST-like sequences can be identified using the PEST search program. For example, the PEST-like sequence can be a region rich in proline (P), glutamic acid (E), serine (S), and threonine (T) residues. Optionally, the PEST-like sequence can be flanked by one or more clusters containing several positively charged amino acids. For example, a PEST-like sequence can be defined as having a higher proline (P), aspartate (D), glutamate (E), serine (S), and/or threonine ( The length of the T) local concentration is a hydrophilic extension of at least 12 amino acids. In some cases, the PEST-like sequence does not contain a positively charged amino acid, namely arginine (R), histidine (H), and lysine (K). Some PEST-like sequences may contain one or more internal phosphorylation sites, and phosphorylation at these sites precedes protein degradation. [0077] In one example, the PEST-like sequence fits the algorithm disclosed in Rogers et al. In another example, the PEST-like sequence fits the algorithm disclosed in Rechsteiner and Rogers. PEST-like sequences can also be identified by initially scanning positively charged amino acids R, H, and K within a given protein sequence. The total amino acid count between the positively charged side-linking sequences is counted, and further only those motifs containing a plurality of amino acids equal to or higher than the window size parameter are considered. Optionally, the PEST-like sequence must contain at least one P, at least one D or E, and at least one S or T. [0078] The quality of the PEST motif is optimized by means of a scoring parameter based on local enrichment of the key amino acids and hydrophobicity of the motif. The enrichment of D, E, P, S, and T is expressed in mass percent (w/w) and is corrected for 1 equivalent of D or E, 1 equivalent of P, and 1 equivalent of S or T. The calculation of hydrophobicity can also be followed in principle by Kyte and Doolittle (1982)J. Mol. Biol. The method of 157:105 is hereby incorporated by reference in its entirety for all purposes. To simplify the calculation, the Kyte-Doolittle hydrophilicity index, initially between arginine-4.5 to isoleucine + 4.5, was converted to a positive integer using the following linear transformation, which gave the value of arginine 0 to isoleucine 90. : Hydrophilic index = 10 × Kyte-Doolittle hydrophilicity index + 45. The hydrophobicity of the latent PEST motif can also be calculated as the sum of the product of the percentage of moles of each amino acid species and the hydrophobicity index. The desired PEST score is obtained as a combination of the local enrichment term and the hydrophobicity term as indicated by the following equation: PEST score = 0.55 × DEPST - 0.5 × hydrophobicity index. Thus, a PEST-containing peptide can refer to a peptide having a score of at least +5 using the algorithm above. Alternatively, it may mean a score of at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, Peptides of at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 35, at least 38, at least 40 or at least 45. Any other available methods or algorithms known in the art can also be used to identify PEST-like sequences. See, for example, CaS Predictor (Garay-Malpartida et al. (2005)Bioinformatics 21 Supplement 1: i169-76, which is hereby incorporated by reference in its entirety for all purposes. Another method that can be used is as follows: each extension of the appropriate length is calculated by assigning a value of 1 to the amino acid Ser, Thr, Pro, Glu, Asp, Asn or Gln (eg an extension of 30-35 amino acids) Department) PEST index. The coefficient value (CV) of each of the PEST residues is 1, and the CV of each of the other AA (non-PEST) is zero. Examples of PEST-like amino acid sequences are those set forth in SEQ ID NOs: 43-51. An example of a PEST-like sequence is KENSISSMAPPASPPASPKTPIEKKHADEIDK (SEQ ID NO: 43). Another example of a PEST-like sequence is KENSISSMAPPASPPASPK (SEQ ID NO: 44). However, any PEST or PEST-like amino acid sequence can be used. PEST sequence peptides are known and are described, for example, in US 7,635,479; US 7,665,238; and US 2014/0186387, each of which is hereby incorporated by reference in its entirety for all purposes. [0083] The PEST-like sequence may be from a Listeria species, such as from Listeria monocytogenes. For example, the Listeria monocytogenes ActA protein contains at least four such sequences (SEQ ID NOS: 45-48), any of which are suitable for use in the compositions and methods disclosed herein. Other PEST-like sequences include SEQ ID NOS: 52-54. From Streptococcus (Streptococcus Sp. streptolysin O also contains a PEST sequence. For example, Streptococcus pyogenes (Streptococcus pyogenes Streptococcal O contains the PEST sequence KQNTASTETTTTNEQPK (SEQ ID NO: 49) at amino acid 35-51, and S. equi.Streptococcus equisimilis Streptococcal O contains the PEST-like sequence KQNTANTETTTTNEQPK (SEQ ID NO: 50) at amino acid 38-54. Another example of a PEST-like sequence is derived fromLso Genetically encoded Listeria monocytogenesListeria seeligeri ) lysin: RSEVTISPAETPESPPATP (e.g., SEQ ID NO: 51). Alternatively, the PEST-like sequence may be derived from other prokaryotic organisms. Other prokaryotic organisms in which the PEST-like amino acid sequence will be contemplated include, for example, other Listeria species. (1) Listeria lysin O (LLO) An example of a PEST-containing peptide useful in the compositions and methods disclosed herein is a Listeria lysin O (LLO) peptide. An example of an LLO protein is a protein designated GenBank Accession No. P13128 (SEQ ID NO: 55; nucleic acid sequence set forth in GenBank Accession No. X15127). SEQ ID NO: 55 is the protein prior to inclusion of the signal sequence. The first 25 amino acids of the proprotein are signal sequences and are cleaved from LLO when secreted by the bacteria, thereby producing a full length active LLO protein of 504 amino acids without a signal sequence. The LLO peptides disclosed herein may comprise a signal sequence or may comprise a peptide that does not comprise a signal sequence. An exemplary LLO protein that can be used comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 55, or a homolog, variant, isoform of SEQ ID NO: 55, Analogs, fragments, homolog fragments, variant fragments, analog fragments, and isoform fragments. Any sequence encoding a homologue, variant, isoform, analog, homolog fragment, variant fragment or analog fragment of an LLO protein fragment or LLO protein can be used. The sequence identity of the homologous LLO egg to the reference LLO protein can be, for example, greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%. , 93%, 95%, 96%, 97%, 98% or 99%. Another example of an LLO protein is set forth in SEQ ID NO:56. A LLO protein that can be used can comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 56, or a homologue, variant, isoform, or similar of SEQ ID NO: 56. Substances, fragments, homolog fragments, variant fragments, analog fragments and isoform fragments. Another example of a LLO protein is the LLO protein from the Listeria monocytogenes 10403S strain as set forth in the GenBank Accession Number: ZP_01942330 or EBA21833, as described in the GenBank Accession Numbers below. Code: NZ_AARZ01000015 or AARZ01000015.1. Another example of an LLO protein is the LLO protein from the Listeria monocytogenes 4b F2365 strain (See for example , GenBank deposit number: YP_012823), EGD-e strain (See for example , GenBank accession number NP_463733) or any other strain of Listeria monocytogenes. Yet another example of an LLO protein is the LLO protein from the Flavobacterium bacterium HTCC2170 (see, for example, GenBank Accession No.: ZP_01106747 or EAR01433, or encoded by GenBank Accession Number: NZ_AAOC01000003). The LLO protein that can be used may comprise, consist essentially of, or consist of: a homologue, a variant, an isoform, an analog, a fragment, a homologue fragment of the above LLO protein or the above LLO protein. Any of a variant fragment, an analog fragment, and an isoform fragment. [0088] Proteins homologous to LLO or homologs, variants, isoforms, analogs, fragments, homolog fragments, variant fragments, analog fragments and isoform fragments can also be used. One such example is alveolysin, which can be found, for example, in Bacillus hominis (see, eg, GenBank Accession Number: P23564 or AAA22224, or by GenBank Accession Number: M62709). Other such homologous proteins are known. [0089] The LLO peptide can be a full length LLO protein or a truncated LLO protein or LLO fragment. Likewise, the LLO peptide can be an LLO peptide that retains one or more of the native LLO proteins or lacks one or more of the functionality of the native LLO protein. For example, the retained LLO functionality may be to allow bacteria (eg, Listeria) to escape from phagosomes or phagocytic lysosomes, or to enhance the immunogenicity of peptides fused thereto. The functionality retained may also be hemolysis or antigenic. Alternatively, the LLO peptide can be a non-hemolytic LLO. Other functions of LLO are known, and methods and assays for assessing LLO functionality are also known. [0090] The LLO fragment can be a PEST-like sequence or can comprise a PEST-like sequence. The LLO fragment can include one or more of an internal deletion, a C-terminal truncation, and a N-terminal truncation. In some cases, an LLO fragment can contain more than one internal deletion. Other LLO peptides can be full length LLO proteins with one or more mutations. [0091] The hemolytic activity of some LLO proteins or fragments is reduced relative to wild-type LLO, or is a non-hemolytic fragment. For example, an LLO protein can be rendered non-hemolytic by deletion or mutation of the activation domain at the carboxy terminus, by deletion or mutation of cysteine 484, or by deletion or mutation at another position. [0092] Other LLO proteins are rendered non-hemolytic by deletion or mutation of a cholesterol binding domain (CBD), as described in detail in US Pat. . Mutations can include, for example, substitutions or deletions. The entire CBD can be mutated, parts of the CBD can be mutated, or specific residues within the CBD can be mutated. For example, the LLO protein can comprise one or more of residues C484, W491 and W492 of the sequence of SEQ ID NO: 55 (eg, C484, W491, W492, C484 and W491, C484 and W492, W491 and W492, or Mutations of all three residues), or mutations of corresponding residues (eg, corresponding cysteine or tryptophan residues) when optimally aligned with SEQ ID NO:55. As an example, a mutant LLO protein can be produced in which residues C484, W491 and W492 of LLO are substituted with alanine residues which will significantly reduce hemolytic activity relative to wild-type LLO. The mutant LLO protein having the C484A, W491A and W492A mutations is referred to as "mutLLO". As another example, a mutant LLO protein having an internal deletion comprising a cholesterol binding domain can be produced. The sequence of the cholesterol binding domain of SEQ ID NO: 55 is set forth in SEQ ID NO:74. For example, the internal deletion can be a deletion of 1-11 amino acids, a deletion of 11-50 amino acids, or longer. Likewise, the mutated region can be 1-11 amino acids, 11-50 amino acids or longer (eg, 1-50, 1-11, 2-11, 3-11, 4-11, 5-11) , 6-11, 7-11, 8-11, 9-11, 10-11, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1 -9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6 , 3-7, 3-8, 3-9, 3-10, 12-50, 11-15, 11-20, 11-25, 11-30, 11-35, 11-40, 11-50, 11 -60, 11-70, 11-80, 11-90, 11-100, 11-150, 15-20, 15-25, 15-30, 15-35, 15-40, 15-50, 15-60 , 15-70, 15-80, 15-90, 15-100, 15-150, 20-25, 20-30, 20-35, 20-40, 20-50, 20-60, 20-70, 20 -80, 20-90, 20-100, 20-150, 30-35, 30-40, 30-60, 30-70, 30-80, 30-90, 30-100 or 30-150 amino acids ). For example, a mutated region consisting of residues 470-500, 470-510 or 480-500 of SEQ ID NO: 55 will result in a deletion sequence comprising the CBD (residues 483-493 of SEQ ID NO: 55). However, the mutated region may also be a fragment of the CBD or may partially overlap with one of the CBDs. For example, the mutated region can consist of residues 470-490, 480-488, 485-490, 486-488, 490-500, or 486-510 of SEQ ID NO:55. For example, a fragment of CBD (residues 484-492) can be replaced by a heterologous sequence that will significantly reduce hemolytic activity relative to wild-type LLO. For example, CBD (ECTGLAWEWWR; SEQ ID NO: 74) can be replaced by a CTL epitope (ESLLMWITQCR; SEQ ID NO: 75) from the antigen NY-ESO-1, which contains HLA from NY-ESO-1 A2 restricted epitope 157-165. The resulting LLO is called "ctLLO". In some mutated LLO proteins, the mutated region can be replaced by a heterologous sequence. For example, the mutated region can be passed through an equal number of heterologous amino acids, a smaller number of heterologous amino acids, or a greater number of amino acids (eg, 1-50, 1-11, 2-11, 3) -11, 4-11, 5-111, 6-111, 7-11, 8-11, 9-11, 10-11, 1-2, 1-3, 1-4, 1-5, 1-6 , 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3 -4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 12-50, 11-15, 11-20, 11-25, 11-30, 11-35 , 11-40, 11-50, 11-60, 11-70, 11-80, 11-90, 11-100, 11-150, 15-20, 15-25, 15-30, 15-35, 15 -40, 15-50, 15-60, 15-70, 15-80, 15-90, 15-100, 15-150, 20-25, 20-30, 20-35, 20-40, 20-50 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 30-35, 30-40, 30-60, 30-70, 30-80, 30-90, 30 -100 or 30-150 amino acids) instead. Other mutated LLO proteins have one or more point mutations (eg, 1 residue, 2 residues, 3 residues, or more than 3 point mutations). The mutated residues can be contiguous or non-adjacent. [0095] In an exemplary embodiment, the LLO peptide can have a deletion in the signal sequence and a mutation or substitution in the CBD. [0096] Some LLO peptides are N-terminal LLO fragments (ie, LLO proteins with C-terminal deletions). Some LLO peptides are at least 494, 489, 492, 493, 500, 505, 510, 515, 520 or 525 amino acids or have a length of 492-528 amino acids. For example, an LLO fragment can be preceded by about 440 or 441 amino acids of the LLO protein (eg, 441 amino acids prior to SEQ ID NO: 55 or 56, or when optimally aligned with SEQ ID NO: 55 or 56) The corresponding fragment of another LLO protein consists of. Other N-terminal LLO fragments may be preceded by 420 amino acids of the LLO protein (eg, 420 amino acids prior to SEQ ID NO: 55 or 56, or another LLO when optimally aligned with SEQ ID NO: 55 or 56) Composition of the corresponding fragment of the protein). Other N-terminal LLO fragments may be from the amino acid 20-442 of the LLO protein (eg, the amino acid 20-442 of SEQ ID NO: 55 or 56, or when optimally aligned with SEQ ID NO: 55 or 56) Another corresponding fragment of the LLO protein). The other N-terminal LLO fragment comprises any ΔLLO that does not have an activation domain comprising cysteine 484, and in particular does not have cysteine 484. For example, an N-terminal LLO fragment can correspond to 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50 or 25 amine groups before the LLO protein. An acid (eg, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50 or 25 amino acids before SEQ ID NO: 55 or 56, Or a corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO: 55 or 56). Preferably, the fragment comprises one or more PEST-like sequences. The LLO fragment and the truncated LLO protein may contain homologous LLO protein residues corresponding to any of the specific amino acid ranges above. The number of residues need not correspond exactly to the number of residues listed above (eg, if the homologous LLO protein has an insertion or deletion relative to the particular LLO protein disclosed herein). Examples of N-terminal LLO fragments include SEQ ID NOS: 57, 58, and 59. The LLO protein that can be used comprises a sequence consisting of, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 57, 58 or 59, or a homologue, variation of SEQ ID NO: 57, 58 or 59 Body, isoforms, analogs, fragments, homolog fragments, variant fragments, analog fragments and isoform fragments. In some compositions and methods, the N-terminal LLO fragment set forth in SEQ ID NO: 59 is used. An example of a nucleic acid encoding an N-terminal LLO fragment set forth in SEQ ID NO: 59 is SEQ ID NO:60. (2) ActA Another example of a PEST-containing peptide useful in the compositions and methods disclosed herein is the ActA peptide. ActA is a surface-related protein and acts as a framework in infected host cells to promote the polymerization, assembly and activation of host actin polymers to facilitate the passage of Listeria monocytogenes across the cytoplasm. Shortly after entering the mammalian cell cytosol, Listeria monocytogenes induces aggregation of host actin filaments and uses the forces generated by actin polymerization to first move within the cell and then move between cells . ActA is responsible for mediating actin nucleation and actin-based activity. The ActA protein provides multiple binding sites for the host cytoskeleton assembly, thereby acting as a framework to assemble cellular actin polymerization mechanisms. The N-terminus of ActA binds to monomeric actin and acts as a constitutively active nucleation promoting factor by stimulating the intrinsic actin nucleating activity.actA andHly Both genes are members of a 10 kb gene cluster regulated by the transcriptional activator PrfA, andactA It is up-regulated approximately 226 times in mammalian cytosol. Any sequence encoding a homologue, variant, isoform, analog, homolog fragment, variant fragment or analog fragment of the ActA protein or ActA protein can be used. The sequence identity of the homologous ActA protein to the reference ActA protein can be, for example, greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%. , 93%, 95%, 96%, 97%, 98% or 99%. An example of an ActA protein comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:61. Another example of an ActA protein comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:62. The 29 amino acids preceding the proprotein corresponding to any of these sequences are signal sequences and are cleaved from the ActA protein when secreted by the bacteria. The ActA peptide may comprise a signal sequence (eg, amino acid 1-29 of SEQ ID NO: 61 or 62), or may comprise a peptide that does not include a signal sequence. Other examples of ActA proteins comprise, consist essentially of, or consist of: homologs, variants, isoforms, analogs, fragments, homolog fragments, isoforms of SEQ ID NO: 61 or 62 A fragment of an isoform or an analog fragment. [0099] Another example of ActA protein is ActA protein from: Listeria monocytogenes 10403S strain (GenBank accession number: DQ054585), NICPBP 54002 strain (GenBank accession number: EU394959), S3 strain (GenBank deposit) No.: EU394960), NCTC 5348 strain (GenBank accession number: EU394961), NICPBP 54006 strain (GenBank accession number: EU394962), M7 strain (GenBank accession number: EU394963), S19 strain (GenBank accession number: EU394964) or any other single Listeria monocytogenes strain. The LLO protein that can be used may comprise, consist essentially of, or consist of: a homologue, a variant, an isoform, an analog, a fragment, a homologue fragment of the above LLO protein or the above LLO protein. Any of a variant fragment, an analog fragment, and an isoform fragment. The ActA peptide may be a full-length ActA protein or a truncated ActA protein or an ActA fragment (eg, an N-terminal ActA fragment in which the C-terminal portion is removed). Preferably, the truncated ActA protein comprises at least one PEST sequence (eg, more than one PEST sequence). In addition, the truncated ActA protein may optionally comprise an ActA signal peptide. Examples of the PEST-like sequence contained in the truncated ActA protein include SEQ ID NOS: 45-48. Some such truncated ActA proteins comprise at least two of the PEST-like sequences set forth in SEQ ID NOs: 45-48, or a homolog thereof, at least three of the PEST-like sequences set forth in SEQ ID NOs: 45-48 Or a homolog thereof, or all four of the PEST-like sequences set forth in SEQ ID NOS: 45-48 or homologs thereof. Examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of: about residues 30-122, about residues 30-229 of the full length ActA protein sequence (eg, SEQ ID NO: 62), About 30-332 residues, about 30-200 residues or about 30-399 residues. Other examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of: about 50, 100, 150, 200, 233, 250 before the full length ActA protein sequence (eg, SEQ ID NO: 62), 300, 390, 400 or 418 residues. Other examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of: about residues 200-300 or residues 300-400 of the full length ActA protein sequence (e.g., SEQ ID NO: 62). For example, truncated ActA consists of 390 amino acids prior to the wild-type ActA protein as described in US 7,655,238, which is incorporated herein by reference in its entirety for all purposes. As another example, the truncated ActA can be ActA-N100 or a modified version thereof (referred to as ActA-N100*) in which the PEST motif has been deleted and contains a non-conservative QDNKR (SEQ ID NO: 73) substitution, such as US As described in 2014/0186387, the entire contents of this application are hereby incorporated by reference in its entirety for all purposes. Alternatively, the truncated ActA protein may contain residues corresponding to the homologous ActA protein of the amino acid range of one of the above amino acid ranges or any of the ActA peptides disclosed herein. The number of residues need not correspond exactly to the number of residues listed herein (eg, if the homologous ActA protein has an insertion or deletion relative to the ActA protein used herein, the number of residues can be adjusted accordingly). Examples of truncated ActA proteins include, for example, proteins comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 63, 64, 65 or 66 or SEQ ID NO: 63, 64 , 65 or 66 homologs, variants, isoforms, analogs, variant fragments, isoform fragments or analog fragments. SEQ ID NO: 63 is referred to as ActA/PEST1 and consists of the amino acid 30-122 of the full length ActA sequence set forth in SEQ ID NO:62. SEQ ID NO: 64 consists of amino acid 30-229, referred to as ActA/PEST2 or LA229, and consists of the full length ActA sequence set forth in the full length ActA sequence set forth in SEQ ID NO:62. SEQ ID NO: 65 is referred to as ActA/PEST3 and consists of the amino acid 30-332 of the full length ActA sequence set forth in SEQ ID NO:62. SEQ ID NO: 66 is referred to as ActA/PEST4 and consists of the amino acid 30-399 of the full length ActA sequence set forth in SEQ ID NO:62. As a specific example, a truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 64 can be used. Examples of truncated ActA proteins include, for example, proteins comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 67, 69, 70 or 72 or SEQ ID NO: 67, 69 , 70 or 72 homologs, variants, isoforms, analogs, variant fragments, isoform fragments or analog fragments. As a specific example, a truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 67 (encoded by the nucleic acid set forth in SEQ ID NO: 68) can be used. As another specific example, a truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 70 (encoded by the nucleic acid set forth in SEQ ID NO: 71) can be used. SEQ ID NO: 71 is the codeListeria monocytogenes The first 1170 nucleotides of ActA in the 10403S strain. In some cases, an ActA fragment can be fused to a heterologous signal peptide. For example, SEQ ID NO: 72 sets forth an ActA fragment fused to a Hly signal peptide. C. Generation of Immunotherapeutic Constructs Encoding Recombinant Fusion Polypeptides [0103] Also provided herein are methods for producing immunotherapeutic constructs encoding the recombinant fusion polypeptides disclosed herein or compositions comprising the recombinant fusion polypeptides disclosed herein. For example, such methods can include selecting and designing an antigenic peptide for inclusion in an immunotherapeutic construct (and, for example, testing the hydrophilicity of each antigenic peptide and modifying it if its fraction is above a selected hydrophilicity index threshold) Alternatively or not, the antigenic peptide is designed, one or more fusion polypeptides comprising each of the selected antigenic peptides are designed, and a nucleic acid construct encoding the fusion polypeptide is produced. Antigenic peptides can be screened for hydrophobicity or hydrophilicity. Whether the antigenic peptide is hydrophilic or whether its fraction meets or falls below a certain hydrophilicity threshold can be selected, which predicts the secretagability in a particular bacterium of interest, such as Listeria monocytogenes. For example, the antigenic peptide can be scored by the Kyte and Doolittle hydrophilicity index with a 21 amino acid window, and all scores above the cutoff (approximately 1.6) can be excluded because it is unlikely to be mononuclear Balloon-expressing Listeria secretion.See for example , Kyte-Doolittle (1982)J Mol Biol 157(1): 105--132; the subject is hereby incorporated by reference in its entirety for all purposes. Alternatively, the antigenic peptide scored for the selected cutoff value can be altered (eg, changing the length of the antigenic peptide). Other sliding window sizes that may be used include, for example, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or more than 27 amino acids. For example, the sliding window size can be 9-11 amino acids, 11-13 amino acids, 13-15 amino acids, 15-17 amino acids, 17-19 amino acids, 19 - 21 amino acids, 21-23 amino acids, 23-25 amino acids or 25-27 amino acids. Other cutoff values that may be used include, for example, the following ranges 1.2-1.4, 1.4-1.6, 1.6-1.8, 1.8-2.0, 2.0-2.2 2.2-2.5, 2.5-3.0, 3.0-3.5, 3.5-4.0, or 4.0-4.5, or The cutoff value can be 1.4, 1.5, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.3, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. , 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4 or 4.5. The cutoff value can vary, for example, depending on the genus or species of the bacteria that are being used to deliver the fusion polypeptide. [0105] Other suitable hydrophilicity curves or other suitable scales include, for example, those reported below: Rose et al. (1993)Annu Rev Biomol Struct 22:381-415; Biswas et al. (2003)Journal of Chromatography A 1000: 637-655; Eisenberg (1984)Ann Rev Biochem 53:595-623; Abraham and Leo (1987)Proteins : Structure, Function and Genetics 2:130-152; Sweet and Eisenberg (1983)Mol Biol 171:479-488; Bull and Breese (1974)Arch Biochem Biophys 161:665-670; Guy (1985)Biophys J 47:61-70; Miyazawa et al. (1985)Macromolecules 18:534-552; Roseman (1988)J Mol Biol 200:513-522; Wolfenden et al. (1981)Biochemistry 20: 849-855; Wilson (1981)Biochem J 199:31-41; Cowan and Whittaker (1990)Peptide Research 3:75-80; Aboderin (1971)Int J Biochem 2:537-544; Eisenberg et al. (1984)J Mol Biol 179: 125-142; Hopp and Woods (1981)Proc Natl Acad Sci USA 78:3824-3828; Manavalan and Ponnuswamy (1978)Nature 275: 673-674; Black and Mould (1991)Anal Biochem 193:72-82; Fauchere and Pliska (1983)Eur J Med Chem 18:369-375; Janin (1979)Nature 277:491-492; Rao and Argos (1986)Biochim Biophys Acta 869:197-214; Tanford (1962)Am Chem Soc 84:4240-4274; Welling et al. (1985)FEBS Lett 188:215-218; Parker et al. (1986)Biochemistry 25:5425-5431; and Cowan and Whittaker (1990)Peptide Research 3:75-80, each of which is incorporated herein by reference in its entirety for all purposes. [0106] The ability of the antigenic peptide to be labeled with the human leukocyte antigen (HLA) type can be scored (eg, by using an immunological epitope database available at www.iedb.org (Immune). Epitope Database; IED), which includes netMHCpan, ANN, SMMPMBEC, SMM, CombLib_Sidney2008, PickPocket, and netMHCcons, and is rated by the optimal MHC binding of each antigenic peptide. Other sources include TEpredict (tepredict.sourceforge.net/help.html) or other available MHC binding volume measurement tables. Such as Salmonella (Salmonella The cutoff values of the different performance carriers may vary. Optionally, the antigenic peptide can be screened for an immunosuppressive epitope (eg, a T-reg epitope, an IL-10-induced T helper epitope, etc.) to select no antigenic peptide or to avoid immunosuppressive effects. [0108] Depending on the case, a prediction algorithm for epitope immunogenicity can be used to screen for antigenic peptides. However, these algorithms predict which peptide will produce T cell response with an accuracy of up to 20%. Alternatively, no filtering/prediction algorithms are used. Alternatively, antigenic peptides can be screened for immunogenicity. For example, this can involve contacting one or more T cells with an antigenic peptide, and analyzing the immunogenic T cell response, wherein the immunogenic T cell response identifies the peptide as an immunogenic peptide. This may also comprise, after contacting one or more T cells with the peptide, using an immunogenicity assay to measure secretion or measurement of at least one of CD25, CD44 or CD69 selected from the group consisting of comprising IFN-[gamma], TNF-[alpha]. Secretion of cytokines, a population of IL-1 and IL-2, wherein increased secretion identifies the peptide as comprising one or more T cell epitopes. The selected antigenic peptides can be arranged in one or more candidate sequences for potential fusion polypeptides. If there are more antigenic peptides than can be placed in a single plastid, the priority sequence of the different antigenic peptides can be specified/different and/or divided into different fusion polypeptides (eg, for inclusion in different recombinant Listerias) In the strain of the genus). The priority level can be determined by factors such as relative size, transcriptional priority, and/or overall hydrophobicity of the translated polypeptide. The antigenic peptides can be arranged such that they are directly joined together without any linker or any linker combination between any number of pairs of antigenic peptides, as disclosed in more detail elsewhere herein. The number of linear antigenic peptides to be included can be determined based on the following considerations: the number of desired constructs versus the mutation load, the translation and secretion efficiency of multiple epitopes from a single plastid, and the bacteria containing the plastid orLm The required MOI. The hydrophobicity of the combination of antigenic peptides or the entire fusion polypeptide (ie, comprising the antigenic peptide and PEST-containing peptide and any label) is also scored. For example, the hydrophilicity of all fusion antigen peptides or the entire fusion polypeptide can be scored by the Kyte and Doolittle hydrophilicity index with a 21 amino acid sliding window. If any of the region scores is above the cutoff value (eg, about 1.6), the antigenic peptides can be reordered or shuffled within the fusion polypeptide until an acceptable sequence of antigenic peptides is found (ie, the order in which the no region score is above the cutoff value) until. Alternatively, any problematic antigenic peptides can be removed or redesigned to different sizes. Alternatively or additionally, one or more linkers between antigenic peptides as disclosed elsewhere herein may be added or modified to alter hydrophobicity. As with the hydrophilicity test for individual antigenic peptides, other window sizes can be used, or other cutoff values can be used (eg, depending on the genus or species of the bacteria that are being used to deliver the fusion polypeptide). In addition, other suitable hydrophilicity curves or other suitable scales can be used. Optionally, the combination of antigenic peptides or the entire fusion polypeptide can be further screened for immunosuppressive epitopes (eg, T-reg epitopes, IL-10 induced T helper epitopes, etc.) to select no antigen Peptides or avoid immunosuppressive effects. Nucleic acids encoding candidate antigenic peptide combinations or fusion polypeptides can then be designed and optimized. For example, the sequences can be optimized to achieve a level of translation, duration of expression, level of secretion, level of transcription, and any combination thereof. For example, the increase can be from 2 to 1000 times, from 2 to 500 times, from 2 to 100 times, from 2 to 50 times, from 2 to 20 times, 2 times to the non-optimized sequence of the control group. 10 fold or 3 fold to 5 fold [0113] For example, a fusion polypeptide or a nucleic acid encoding the fusion polypeptide can be optimized to achieve a reduction in the level of secondary structure that may be formed in the oligonucleotide sequence, or alternatively Optimized to prevent enzyme attachment of any modifiable sequence. For example, expression in bacterial cells can be achieved by transcriptional silence, low mRNA half-life, secondary structure formation, attachment sites for oligonucleotide binding molecules such as suppressors and repressors, and the use of rare tRNA pools. Blocked. The source of many problems in bacterial expression is found within the original sequence. Optimization of RNA may include modification of the cis-acting element, modification of its GC content, alteration of codon bias with respect to non-limiting tRNA pools of bacterial cells, and avoidance of internal homologous regions. Therefore, optimizing the sequence may be accompanied by, for example, adjusting regions having very high (&gt; 80%) or very low (&lt; 30%) GC content. Optimizing the sequence may be accompanied by, for example, one or more of the sequence motifs that avoid the following cis-acting: internal TATA匣, purine and ribosome entry sites; AT-rich or GC-rich sequence extensions Repetitive sequence and RNA secondary structure; (hidden) splice donor and acceptor sites; branching points; or a combination thereof. Optimizing performance may also be accompanied by the addition of sequence elements to the flanking regions of the gene and/or elsewhere in the plastid. Optimizing the sequence may also be accompanied by modification of the codon usage, eg, for codon biases of the host gene (eg, the Listeria monocytogenes gene). For example, the following codons can be used for Listeria monocytogenes.A nucleic acid encoding a fusion polypeptide can be produced and introduced into a delivery vehicle such as a bacterial strain or a Listeria strain. Other delivery vehicles may be suitable for DNA immunotherapy or peptide immunotherapy, such as acne virus or virus like particles. Once the plastid encoding the fusion polypeptide is produced and introduced into a bacterial strain or a Listeria strain, the bacterial or Listeria strain can be cultured and characterized to confirm the expression and secretion of the fusion polypeptide comprising the antigenic peptide. III. Recombinant Bacteria or Listeria Strains [0116] Also provided herein are recombinant bacterial strains, such as Listeria strains, comprising a recombinant fusion polypeptide disclosed herein or encoding the recombinant fusion polypeptide as disclosed elsewhere herein. Nucleic acid. Preferably, the bacterial strain is a Listeria strain, such as Listeria monocytogenes (Lm ) strain.Lm There are a number of inherent advantages as a vaccine carrier. Bacteria grow extremely efficiently in vitro without special requirements, and they lack LPS, which is a major virulence factor in Gram-negative bacteria, such as Salmonella. Since the gene attenuated Lm vector can be easily eliminated by antibiotics in the case of serious adverse effects, and unlike some viral vectors, the integration of genetic material into the host genome does not occur, so the gene attenuated Lm vector also provides additional safety. [0117] The recombinant Listeria strain can be any Listeria strain. Examples of suitable Listeria strains include Listeria monocytogenes and Listeria monocytogenes (Listeria grayi ) Listeria monocytogenesListeria ivanovii ) Listeria monocytogenesListeria murrayi ) Listeria monocytogenesListeria welshimeri L. monocytogenesLm Or any other Listeria species known in the art. Preferably, the recombinant Listeria strain is a strain of the species Listeria monocytogenes. Examples of Listeria monocytogenes strains include the following:10403S Wild type Listeria monocytogenes (see, for example, Bishop and Hinrichs (1987)J Immunol 139:2005-2009; Lauer et al. (2002)J Bact 184:4177-4186);Listeria monocytogenes DP-L4056, which is cured by phage (see, for example, Lauer et al. (2002)J Bact 184:4177-4186); Listeria monocytogenes DP-L4027, which is recovered by phage and hasHly Gene deletion (see, for example, Lauer et al. (2002)J Bact 184:4177- 4186; Jones and Portnoy (1994)Infect Immunity 65:5608-5613); Listeria monocytogenes DP-L4029, which is recovered by phage and hasactA Gene deletion (see, for example, Lauer et al. (2002)J Bact 184:4177-4186; Skoble et al. (2000)J Cell Biol 150: 527- 538); Listeria monocytogenes DP-L4042 (ΔPEST) (see, for example, Brockstedt et al. (2004)Proc Natl Acad Sci. USA 101:13832-13837 and supporting information); Listeria monocytogenes DP-L4097 (LLO-S44A) (see, for example, Brockstedt et al. (2004)Proc Natl Acad Sci USA 101:13832-13837 and support information); Listeria monocytogenes DP-L4364 (ΔlplA Lipoic acid protein ligase) (see, for example, Brockstedt et al. (2004)Proc Natl Acad Sci USA 101:13832-13837 and support information); Listeria monocytogenes DP-L4405 (ΔinlA (See, for example, Brockstedt et al. (2004)Proc Natl Acad Sci USA 101:13832-13837 and support information); Listeria monocytogenes DP-L4406 (ΔinlB (See, for example, Brockstedt et al. (2004)Proc Natl Acad Sci USA 101:13832-13837 and support information); Listeria monocytogenes CS-LOOOl (ΔactA ;ΔinlB (See, for example, Brockstedt et al. (2004)Proc Natl Acad Sci USA 101:13832-13837 and support information); Listeria monocytogenes CS-L0002 (ΔactA ;ΔlplA (See, for example, Brockstedt et al. (2004)Proc Natl Acad Sci USA 101:13832-13837 and support information); Listeria monocytogenes CS-L0003 (LLO L461T; ΔlplA (See, for example, Brockstedt et al. (2004)Proc Natl Acad Sci USA 101:13832-13837 and support information); Listeria monocytogenes DP-L4038 (ΔactA ;LLO L461T) (See, for example, Brockstedt et al. (2004)Proc Natl Acad Sci USA 101:13832-13837 and supporting information); Listeria monocytogenes DP-L4384 (LLO S44A; LLO L461T) (see, for example, Brockstedt et al. (2004)Proc Natl Acad Sci USA 101:13832-13837 and support information);lplA1 Missing Listeria monocytogenes strain (encoding lipoic acid protein ligase LplA1) (see, for example, O'Riordan et al. (2003)Science 302: 462-464); Listeria monocytogenes DP-L4017 (with 10403S of LLO L461T) (see for example, US 7,691,393); Listeria monocytogenes EGD (see, for example, GenBank accession number AL591824) ). In another specific example, the Listeria strain is Listeria monocytogenes EGD-e (see GenBank accession number NC_003210; ATCC accession number BAA-679); Listeria monocytogenes DP-L4029 (actA Missing, depending on the situationuvrAB Deletion combination (DP-L4029uvrAB) (see for example, US 7,691,393); Listeria monocytogenesactA -/inlB - double mutant (see, for example, ATCC Accession No. PTA-5562); Listeria monocytogeneslplA Mutant orHly Mutant (see, for example, US 2004/0013690); Listeria monocytogenesDal /Dat Double mutant (see for example, US 2005/0048081). Other Listeria monocytogenes strains include those strains that have been modified (eg, by plastid and/or by genomic integration) to contain nucleic acids encoding one or any combination of the following genes:Hly (LLO; Listeria lysin);Iap (p60);inlA ;inlB ;inlC ;Dal (alanine racemase);Dat (D-amino acid transaminase);plcA ;plcB ;actA Or mediate the growth, spreading, rupture of a single-walled vesicle, cleavage of a bivalve vesicle, binding to a host cell or any nucleic acid that is taken up by the host cell. Each of the above references is hereby incorporated by reference in its entirety for all purposes. The recombinant bacterium or Listeria may have wild-type pathogenicity, may have attenuated pathogenicity or may be non-pathogenic. For example, recombinant Listeria can be sufficiently toxic to escape phagosomes or phagocytose lysosomes and enter cytosol. Such Listeria strains may also be live attenuated Listeria strains comprising at least one attenuating mutation, deletion or inactivation as disclosed elsewhere herein. Preferably, the recombinant Listeria is an attenuated auxotrophic strain. An auxotrophic strain is a strain that is unable to synthesize a specific organic compound required for its growth. Examples of such strains are described in US Pat. No. 8,114,414, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. Preferably, the recombinant Listeria strain lacks an antibiotic resistance gene. For example, such recombinant Listeria strains may comprise plastids that do not encode an antibiotic resistance gene. However, some of the recombinant Listeria strains provided herein comprise a plastid comprising a nucleic acid encoding an antibiotic resistance gene. Antibiotic resistance genes can be used in conventional selection and selection methods commonly used in molecular biology and vaccine preparation. Exemplary antibiotic resistance genes include gene products that confer resistance to: ampicillin, penicillin, methicillin, streptomycin, erythromycin, kanamycin, tetracycline, chloramphenicol (CAT), Neomycin, hygromycin and gentamicin. A. A bacterial or Listeria strain comprising a recombinant fusion polypeptide or a nucleic acid encoding a recombinant fusion polypeptide. The recombinant bacterial strain (e.g., a Listeria strain) disclosed herein comprises a recombinant fusion polypeptide disclosed herein or as A nucleic acid encoding the recombinant fusion polypeptide disclosed elsewhere herein. In a bacterial or Listeria strain comprising a nucleic acid encoding a recombinant fusion protein, the nucleic acid can be codon-optimized. An example of the best codon used by Listeria monocytogenes for each amino acid is shown in US 2007/0207170, which is hereby incorporated by reference in its entirety for all purposes. A nucleic acid is codon-optimized if at least one codon in the nucleic acid is replaced by a codon that is more frequently used by the Listeria monocytogenes than the codon in the original sequence. The nucleic acid may be present in an episomal plastid within the bacterial or Listeria strain, and/or the nucleic acid may be integrated into the bacterial or Listeria strain via the genome. Some recombinant bacteria or Listeria strains comprise two separate nucleic acids encoding two recombinant fusion polypeptides as disclosed herein: one nucleic acid is in an episomal plastid, and one nucleic acid is genomically integrated into a bacterial or Listeria strain in. The additional plastid may be a plastid that is stably maintained in vitro (in cell culture), in vivo (in the host), or in vitro and in vivo. If in an episomal, the open reading frame encoding the recombinant fusion polypeptide can be operably linked to a promoter/regulatory sequence in the plastid. If the genome is integrated into a bacterial or Listeria strain, the open reading frame encoding the recombinant fusion polypeptide can be operably linked to an exogenous promoter/regulatory sequence or operably linked to an endogenous promoter/ Adjust the sequence. Examples of promoter/regulatory sequences suitable for driving the constitutive expression of genes are well known and include, for example, ListeriaHly ,hlyA ,actA ,prfA ,andP60 Promoter, streptococcusBac Promoter, Streptomyces griseusStreptomyces griseus )sgiA Promoter and Bacillus thuringiensis (B. thuringiensis )phaZ Promoter. In some cases, the inserted gene of interest is uninterrupted or subject to regulatory constraints that often occur when integrated into genomic DNA, and in some cases, the presence of the inserted heterologous gene does not cause rearrangement or disruption of important regions of the cell itself. . Such a recombinant bacterium or Listeria strain can be obtained by using a plastid or vector comprising a nucleic acid encoding a recombinant fusion polypeptide for a bacterium or Listeria strain or attenuated bacterium or Liss described elsewhere herein. The strain of the genus of the genus is transformed to manufacture. The plastid may be an additional plastid that does not integrate into the host chromosome. Alternatively, the plastid may be an integrated plastid that is integrated into the chromosome of a bacterial or Listeria strain. The plastids used herein may also be multiple complexes. Methods for transforming bacteria are well known and include methods based on calcium chloride competent cells, electroporation methods, phage-mediated transduction, chemical transformation techniques, and physical transformation techniques. See, for example, de Boer et al. (1989)Cell 56:641-649; Miller et al. (1995)FASEB J. 9: 190-199; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al. (1997) Current Protocols in Molecular Biology, John Wiley & Sons, New York; Gerhardt et al. Edited, 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, DC; and Miller, 1992, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, each of which It is incorporated herein by reference in its entirety for all purposes. A bacterial or Listeria strain having a genomically integrated heterologous nucleic acid can be produced, for example, by using a site-specific integration vector in which homologous recombination is used to produce a bacterium or Listeria comprising the integrated gene. The integration vector can be any site-specific integration vector capable of infecting a bacterial or Listeria strain. Such integration vectors may comprise, for example, a PSA attPP' site, a gene encoding a PSA integrase, a U153 attPP' site, a gene encoding U153 integrase, an A118 attPP' site, a gene encoding A118 integrase, or any other known AttPP' site or any other phage integrase. Such bacteria or Listeria strains comprising the integrated gene can also be used to integrate heterologous nucleic acids into bacteria orListeria Any other known method in the chromosome to produce. Techniques for homologous recombination are well known and described, for example, in Baloglu et al. (2005)Vet Microbiol 109(1-2): 11-17); Jiang et al. 2005)Acta Biochim Biophys Sin (Shanghai) 37(1): 19-24) and US 6,855,320, each of which is incorporated herein in its entirety for all purposes. Integration into bacterial or Listeria chromosomes can also be achieved using transposon insertion. Techniques for transposon insertion are well known, and for example the construction of DP-L967 by Sun et al. (1990)Infection and Immunity 58: 3770-3778, which is incorporated herein by reference in its entirety for all purposes. Transposon mutation induction results in stable genomic insertion, but the location of the heterologous nucleic acid inserted into the genome is unknown. Integration into bacterial or Listeria chromosomes can also be achieved using phage integration sites (see, eg, Lauer et al. (2002)J Bacteriol 184(15): 4177-4186, which is hereby incorporated by reference in its entirety for all purposes. ). For example, an integrase gene and attachment site of a bacteriophage (eg, U153 or Listeria phage) can be used to insert a heterologous gene into a corresponding attachment site, which can be any suitable in the genome. Site (for exampleArg tRNA genecomK Or 3' end). Endogenous prophage can be healed from the attachment site utilized prior to integration of the heterologous nucleic acid. Such methods can produce, for example, a single replica integrator. To avoid the "phage curing step", PSA phage based on the phage integration system can be used (see, for example, Lauer et al. (2002)J Bacteriol 184: 4177-4186, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes. Maintaining the integrated gene may require continuous selection, for example by antibiotics. Alternatively, a phage-based chromosomal integration system that does not require antibiotic selection can be established. In fact, an auxotrophic host strain can be supplemented. For example, a phage-based chromosomal integration system for clinical applications can be used in which a host strain that is auxotrophic for an essential enzyme (including, for example, D-alanine racemase) is used (eg,Lm dal (-)Dat (-)). Binding can also be used to introduce genetic material and/or plastid into bacteria. Methods for binding are well known and described, for example, in Nikodinovic et al. (2006)Plasmid 56(3): 223-227 and Auchtung et al. (2005)Proc Natl Acad Sci USA Each of 102 (35): 12554-12559 is hereby incorporated by reference in its entirety for all purposes. [0130] In one embodiment, the recombinant bacterium or Listeria strain may comprise a nucleic acid encoding a recombinant fusion polypeptide that is endogenously integrated into the genome of the bacterium or Listeria by genomeactA Sequence (encoding ActA protein) or endogenousHly The open reading frame of the sequence (encoding the LLO protein). For example, the expression and secretion of a fusion polypeptide can be endogenousactA Under the control of the promoter and ActA signal sequence or may be endogenousHly Under the control of the promoter and LLO signal sequence. As another example, a nucleic acid encoding a recombinant fusion polypeptide can be substituted for the ActA protein.actA Sequence or encode LLO proteinHly sequence. [0131] The selection of the recombinant bacterium or Listeria strain can be achieved by any means. For example, antibiotic selection can be used. Antibiotic resistance genes can be used in conventional selection and selection methods commonly used in molecular biology and vaccine preparation. Exemplary antibiotic resistance genes include gene products that confer resistance to: ampicillin, penicillin, methicillin, streptomycin, erythromycin, kanamycin, tetracycline, chloramphenicol (CAT), Neomycin, hygromycin and gentamicin. Alternatively, an auxotrophic strain can be used, and in addition to or in addition to the antibiotic resistance gene, an exogenous metabolic gene can be used for selection. As an example, in order to select an auxotrophic bacterium comprising a plastid encoding a metabolic enzyme or a supplemental gene provided herein, the transformed auxotrophic bacterium can be directed to a gene encoding a metabolic enzyme (eg, amino acid metabolism) The gene) or the complement of the gene is expressed in a medium selected for growth. Alternatively, the thermosensitive plastids can be used to select recombinants, or any other member known to be used to select recombinants. B. Attenuation of Bacterial or Listeria Strains [0132] The recombinant bacterial strains disclosed herein (eg, recombinant Listeria strains) can be attenuated. The term "attenuated" encompasses the attenuation of the ability of a bacterium to cause disease in a host animal. For example, the pathogenic character of attenuated Listeria strains can be reduced compared to wild type Listeria, but the attenuated Listeria can be grown and maintained in culture. For example, inoculated BALB/c mice intravenously with attenuated Listeria, the lethal dose of 50% vaccinated animals survived (LD₅₀ ) better than the LD of wild type Listeria₅₀ It is at least about 10 times higher, more preferably at least about 100 times, more preferably at least about 1,000 times, even more preferably at least about 10,000 times and most preferably at least about 100,000 times. The attenuated Listeria strain is therefore a strain that does not kill the animal with which it is administered, or only if the number of bacteria administered is significantly greater than the number of wild-type non-attenuated bacteria required to kill the same animal A strain that kills an animal. Attenuated bacteria are also understood to mean bacteria that cannot be replicated in a general environment because there is no nutrients for their growth in this environment. Therefore, bacteria are limited to replication in a controlled environment that provides the required nutrients. Attenuated strains are environmentally safe because they cannot be uncontrolled for replication. (1) Method for attenuating bacteria and Listeria strains [0133] Attenuation can be achieved by any known means. For example, such attenuated strains may lack one or more endogenous pathogenic genes or one or more endogenous metabolic genes. Examples of such genes are disclosed herein, and attenuation can be achieved by inactivating any or any combination of the genes disclosed herein. Inactivation can be achieved, for example, via deletion or via mutation (eg, inactivating mutation). The term "mutation" includes any type of mutation or modification of a sequence (nucleic acid or amino acid sequence) and may encompass deletions, truncations, insertions, substitutions, disruptions or shifts. For example, mutations can include in-frame transfer mutations, mutations that cause premature termination of the protein, or regulatory sequence mutations that affect gene expression. Mutation induction can be achieved using recombinant DNA techniques or using conventional mutation inducing techniques that utilize mutant chemicals or radiation and subsequently select mutants. Deletion mutants may be preferred because of the low probability of reversal. The term "metabolic gene" refers to a gene encoding an enzyme involved in or required for the nutrition utilized or required by the host bacterium. For example, the enzyme may be involved or required for the nutrition required to synthesize the host bacteria for continued growth. The term "pathogenic" gene, including its presence or activity in the genome of an organism, contributes to the pathogenicity of the organism (eg, enables the organism to achieve colonization of the ecological habitat in the host (including attachment to the cell) Genes for immune evasion (evading the host's immune response), immunosuppression (inhibiting the host's immune response), entering and leaving the cell, or obtaining nutrients from the host). A specific example of such an attenuated strain is Listeria monocytogenes (Lm )Dal (-)Dat (-) (Lmdd ). Another example of such an attenuated strain isLm Dal (-)Dat (-)ΔactA (LmddA ). See for example, US 2011/0142791, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.LmddA Due to deletion of endogenous pathogenic genesactA And attenuated Listeria strains. Such strains can be supplemented byDal The gene retains the plastid for antigen expression in vivo and in vitro. Alternatively,LmddA EndogenousDal , Dat ,andactA Mutation in the geneDal/dat/actA Listeria. Such mutations can be, for example, deletions or other inactivating mutations. Another specific example of an attenuated strain isLm prfA (-) or atprfA A strain with a partial deletion or inactivation mutation in the gene. PrfA protein control containsLm The expression of a regulator of the essential pathogenic gene required for colonization of its vertebrate host; thus the prfA mutation strongly impairs the ability of PrfA to activate the expression of a PrfA-dependent toxic gene. Yet another specific example of an attenuated strain isLm inlB (-)actA (-), two key genes in which bacteria are naturally pathogenic, internalizing proteinsB andAct A , is removed. Other examples of attenuated bacteria or Listeria strains include bacteria or Listeria strains that lack one or more endogenous pathogenic genes. Examples of such genes includeListeria In the middleactA ,prfA ,plcB ,plcA ,inlA ,inlB ,inlC ,inlJ andBsh . The attenuated Listeria strain may also be a double mutant or a triple mutant of any of the above mentioned strains. The attenuated Listeria strain may comprise a mutation or deletion of each of the genes provided herein, or comprise, for example, up to ten of any of the genes provided herein (eg, includingactA ,prfA andDal/dat Mutation or deletion of a gene). For example, an attenuated Listeria strain may comprise an endogenous internalizing proteinC (inlC Gene mutation or deletion and/or endogenousactA Mutation or deletion of a gene. Alternatively, the attenuated Listeria strain may comprise an endogenous internalizing proteinB (inlB Gene mutation or deletion and/or endogenousactA Mutation or deletion of a gene. Alternatively, the attenuated Listeria strain may comprise endogenousinlB ,inlC andactA Mutation or deletion of a gene. Lack of endogenousity involved in the processactA Gene and/or endogenousinlC Gene or endogenousinlB Gene suppressionListeria Displacement to adjacent cells, thereby causing an unexpectedly high amount of attenuation, increasing immunogenicity and acting as a strain backbone. Attenuated Listeria strains may also be includedplcA andplcB A double mutant of a mutant or deletion of both. In some cases, the strain can be EGDListeria The main chain is constructed. [0138] The bacterial or Listeria strain may also be an auxotrophic strain having a mutation in a metabolic gene. As an example, a strain may lack one or more endogenous amino acid metabolism genes. For example, Listeria auxotrophic strains that produce, for example, a lack of D-alanine, can be achieved in a variety of ways, including deletion mutations, insertion mutations, in-frame transfer mutations, mutations that cause premature termination of the protein, or Regulatory sequence mutations that affect gene expression. Deletion mutants may be preferred because of the low probability of reversal with auxotrophic phenotypes. As an example, the ability of D-alanine mutants produced according to the protocols presented herein to be grown in the absence of D-alanine can be tested in a simple laboratory culture assay. Those mutants that cannot grow in the absence of this compound can be selected. Examples of the endogenous amino acid metabolism gene include a vitamin synthesis gene, a gene encoding a pantothenate synthase, a D-glutamic acid synthase gene, and a D-alanine transaminase (Dat Gene, D-alanine racemase (Dal )gene,Dga , involved in the synthesis of diaminopimelic acid (DAP), involved in cysteine synthase A (cysK Synthetic gene, vitamin B12-independent methionine synthase,trpA ,trpB ,trpE ,asnB ,gltD ,gltB ,leuA ,argG andthrC . Listeria strains may lack two or more than two such genes (egDat andDal ). D-glutamic acid synthesis is partially affectedDal Gene control, this gene is involved in the conversion and reverse reaction of D-glu + pyr to α-ketoglutarate + D-ala. [0140] As another example, an attenuated Listeria strain may lack an endogenous synthetase gene, such as an amino acid synthesis gene. Examples of such genes includefolP a gene encoding a dihydrouridine synthase family protein,ispD ,ispF a gene encoding phosphoenolpyruvate synthase,hisF ,hisH ,fliI a gene encoding a ribosome larger subunit pseudouridine synthase,ispD a gene encoding a bifunctional GMP synthetase/glutamate transaminase protein,cobS ,cobB ,cbiD a gene encoding uroporphyrin-III C-methyltransferase/uroporphyrinogen-III synthase,cobQ ,uppS ,truB ,Dxs ,mvaS ,dapA ,ispG ,folC a gene encoding a citrate synthase,argJ a gene encoding 3-deoxy-7-phosphonate synthase, a gene encoding a 吲哚-3-glycerol-phosphate synthase, a gene encoding an ortho-aminobenzoate synthase/glutamate transaminase module ,menB a gene encoding a menaquinone-specific iso-branched acid synthase, a gene encoding a phosphoribosylmethionine-synthesizing enzyme I or II, a gene encoding a phosphoribosyl-imidazole-amber-carbamamine synthase,carB ,carA ,thyA ,mgsA ,aroB ,hepB ,rluB ,ilvB ,ilvN ,alsS ,fabF ,fabH a gene encoding a pseudouridine synthase,pyrG ,truA ,pabB And the atp synthase gene (for example,atpC , atpD-2 , aptG , atpA-2 and many more). [0141] Attenuated Listeria strains may lack endogenousphoP ,aroA ,aroC ,aroD orplcB . As yet another example, an attenuated Listeria strain may lack an endogenous peptide transporter. Examples include genes encoding the following: ABC transporter/ATP binding/permease protein, oligopeptide ABC transporter/oligopeptide binding protein, oligopeptide ABC transporter/permease protein, zinc ABC transporter/zinc binding protein, sugar ABC Transporter, phosphate transporter, ZIP zinc transporter,EmrB /QacA Family-resistant drug transporters, sulfate transporters, proton-dependent oligopeptide transporters, magnesium transporters, formate/nitrite transporters, spermidine/putosine ABC transporters, a Na/Pi Transporter, phospho sugar transporter, glutamine ABC transporter, major facilitator family transporter, glycine betaine/L-proline ABC transporter, molybdenum ABC transporter, teichoic acid ABC transporter, Cobalt ABC transporter, ammonium transporter, amino acid ABC transporter, cell division ABC transporter, manganese ABC transporter, iron compound ABC transporter, maltose/maltodextrin ABC transporter,Bcr /CflA A family of drug-resistant transporters and a subunit of one of the above proteins. [0142] Other attenuated bacteria and Listeria strains may be endogenous metabolic enzymes, which are metabolized for bacterial growth processes, replication processes, cell wall synthesis, protein synthesis, fatty acid metabolism, or Amino acid in any other growth or replication process. Similarly, attenuated strains may lack endogenous metabolic enzymes that catalyze the formation of amino acids used in cell wall synthesis, catalyze the synthesis of amino acids used in cell wall synthesis, or may participate in the synthesis of cell wall synthesis. The amino acid used. Alternatively, amino acids can be used for cell wall biogenesis. Alternatively, the metabolic enzyme is a synthetase for D-glutamic acid, a cell wall component. Other attenuated Listeria strains may lack the gene for synthesis by D-glutamic acid,Dga , a metabolic enzyme encoded by the arr (alanine racemase) gene or any other enzyme involved in the synthesis of alanine. Still other examples of metabolic enzymes that can be deficient in Listeria strains include enzymes encoded by:serC (a phosphoserine transaminase),Asd (aspartate beta-halfaldehyde dehydrogenase; involved in the synthesis of the cell wall component diaminopimelate), encoding gsaB-glutamic acid-1-semialdehyde transaminase (catalyzed by (S)-4-amino- a 5-sided oxyvalerate to form a 5-aminoethyl propyl propionate) gene,hemL (catalyzing the formation of 5-aminoethyl propyl propionate from (S)-4-amino-5-side valerate),aspB (catalyzing the formation of oxaloacetate from L-aspartate and 2-oxoethoxyglutarate and the aspartate transaminase of L-glutamate),argF-1 (involved in arginine biosynthesis),aroE (participating in amino acid biosynthesis),aroB (participating in 3-dehydroquinic acid ester biosynthesis),aroD (participating in amino acid biosynthesis),aroC (participating in amino acid biosynthesis),hisB (involved in histidine biosynthesis),hisD (involved in histidine biosynthesis), hisG (in histidine biosynthesis),metX (participating in methionine biosynthesis),proB (participating in proline biosynthesis),argR (involved in arginine biosynthesis),argJ (involved in arginine biosynthesis),Thil (involved in thiamine biosynthesis),LMOf2365_1652 (participating in tryptophan biosynthesis),aroA (participating in tryptophan biosynthesis),ilvD (participating in proline and isoleucine biosynthesis), ilvC (involved in proline and isoleucine biosynthesis), leuA (involved in leucine biosynthesis),dapF (participating in lysine biosynthesis), andthrB (Participating in threonine biosynthesis) (all GenBank accession number NC_002973). The attenuated Listeria strain can be produced by mutation of other metabolic enzymes, such as tRNA synthetase. For example, metabolic enzymes can betrpS The gene encodes a gene encoding a tryptophanyl tRNA synthetase. For example, the host strain bacteria can be Δ (trpS aroA And two markers can be included in the integration vector. Other examples of metabolic enzymes that can be mutated to produce an attenuated Listeria strain include enzymes encoded by:murE (participating in the synthesis of diaminopimelic acid; GenBank accession number: NC_003485), LMOf2365_2494 (participating in the biosynthesis of teichoic acid),WecE (lipopolysaccharide biosynthesis protein rffA; GenBank accession number: AE014075.1) oramiA (An N-acetyl endosome-L-alanine guanamine). Still other examples of metabolic enzymes include aspartate transaminase, histamine-phosphate transaminase (GenBank Accession No. NP_466347) or the cell wall teic acid glycosylated protein GtcA. Other examples of metabolic enzymes that can be mutated to produce an attenuated Listeria strain include a synthetase for a peptidoglycan component or precursor. The component may be, for example, UDP-N-acetylcholine pentapeptide, UDP-N-acetylglucosamine, MurNAc-(pentapeptide)-pyrophosphonium-undecitol, GlcNAc-p-(1, 4) -MurNAc-(pentapeptide)-pyrophosphonate or any other peptidoglycan component or precursor. Still other examples of metabolic enzymes that can be mutated to produce an attenuated Listeria strain include metabolic enzymes encoded by:murG ,murD ,murA-1 ormurA-2 (All are described in GenBank registration number NC_002973). Alternatively, the metabolic enzyme can be any other synthetic enzyme used in the peptidoglycan component or precursor. The metabolic enzyme may also be a transglycosidase, a transpeptidase, a carboxypeptidase, any other class of metabolic enzymes or any other metabolic enzyme. For example, the metabolic enzyme can be any other Listeria metabolic enzyme or any other Listeria monocytogenes metabolic enzyme. Other bacterial strains can be attenuated by mutating the corresponding orthologous genes in the other bacterial strains as described above for Listeria. (2) Method for supplementing attenuated bacteria and Listeria strains [0149] The attenuated bacteria or Listeria strains disclosed herein may further comprise a supplemental gene or a complementary attenuating mutation (eg, supplementation) A nucleic acid of a metabolic enzyme of an auxotrophic strain of a auxotrophic Listeria strain. For example, a nucleic acid having a first open reading frame encoding a fusion polypeptide as disclosed herein can further comprise a second open reading frame comprising a supplemental gene or encoding a supplemental metabolic enzyme. Alternatively, the first nucleic acid can encode a fusion polypeptide and the second nucleic acid alone can comprise a supplemental gene or encode a supplemental metabolic enzyme. [0150] The complement gene may be extrachromosomal or may be integrated into the genome of the bacterium or Listeria. For example, an auxotrophic Listeria strain can comprise an additional plastid comprising a nucleic acid encoding a metabolic enzyme. Such plastids will be contained in Listeria in a free or extrachromosomal manner. Alternatively, the auxotrophic Listeria strain may comprise an integrated plastid (i.e., an integration vector) comprising a nucleic acid encoding a metabolic enzyme. Such integrated plastids can be used to integrate into the Listeria chromosome. Preferably, the episomal or integrative plastid lacks an antibiotic resistance marker. In addition to or in addition to antibiotic resistance genes, metabolic genes can be used for selection. As an example, in order to select an auxotrophic bacterium comprising a plastid encoding a metabolic enzyme or a supplemental gene provided herein, the transformed auxotrophic bacterium can be directed to a gene encoding a metabolic enzyme (eg, amino acid metabolism) The gene) or the complement of the gene is expressed in a medium selected for growth. For example, bacteria that are auxotrophic for D-glutamic acid synthesis can be transformed with a plastid containing a gene for D-glutamic acid synthesis, and auxotrophic bacteria will be present in the absence of D-glutamic acid. The auxotrophic bacteria that grow under the plastid without using plastid transformation or expressing the protein encoding D-glutamic acid synthesis will not grow. Similarly, when transformed and exhibiting a plastid comprising a nucleic acid encoding an amino acid metabolizing enzyme for D-alanine synthesis, the auxotrophic bacteria for D-alanine synthesis will be in the absence of D-propylamine. Growth in the presence of acid. Such methods for making suitable media containing or lacking the necessary growth factors, extenders, amino acids, vitamins, antibiotics, and the like are well known and commercially available. Once an auxotrophic bacterium comprising a plastid encoding a metabolic enzyme or a supplemental gene provided herein has been selected in a suitable medium, the bacterium can be propagated in the presence of a selection pressure. Such propagation may involve the growth of bacteria in a medium free of auxotrophic factors. The presence of a plastid that exhibits a metabolic enzyme or a supplemental gene in an auxotrophic bacterium ensures that the plastid will replicate with the bacterium, thus continuously selecting the bacterium containing the plastid. The production of a bacterial or Listeria strain can be easily scaled up by adjusting the volume of the medium in which the auxotrophic bacteria containing the plastids are grown. In one embodiment, the attenuated strain hasDal andDat a strain that is deleted or inactivated in dal and dat (eg, Listeria monocytogenes)Lm )Dal (-)Dat (-) (Lmdd )orLm dal (-)Dat (-)ΔactA (LmddA )), and the supplemental gene encodes alanine racemase (for example, byDal Gene encoding) or D-amino acid transaminase (for example, byDat Gene coding). An exemplary alanine racemase protein can have the sequence set forth in SEQ ID NO: 76 (encoded by SEQ ID NO: 78; GenBank Accession Number: AF038438) or can be a homologue, variant of SEQ ID NO: 76 , isoforms, analogs, fragments, homolog fragments, variant fragments, analog fragments or isoform fragments. The alanine racemase protein can also be any other Listeria agglutinase protein. Alternatively, the alanine racemase protein can be any other Gram-positive alanine racemase protein or any other alanine racemase protein. An exemplary D-amino acid transaminase protein can have the sequence set forth in SEQ ID NO: 77 (encoded by SEQ ID NO: 79; GenBank Accession Number: AF038439) or can be a homologue of SEQ ID NO: 77 , variants, isoforms, analogs, fragments, homolog fragments, variant fragments, analog fragments or isoform fragments. D-amino acid transaminase protein can also be any otherListeria D-amino acid transaminase protein. Alternatively, the D-amino acid transaminase protein can be any other Gram-positive D-amino acid transaminase protein or any other D-amino acid transaminase protein. [0154] In another embodiment, the attenuated strain hasprfA a strain that is deleted or inactivated in prfA (egLm prfA (-)), and the supplemental gene encodes the PrfA protein. For example, the complement gene can encode a mutant PrfA (D133V) protein that restores a portion of the PrfA function. An example of a wild-type PrfA protein is set forth in SEQ ID NO: 80 (encoded by the nucleic acid set forth in SEQ ID NO: 81), and an example of a D133V mutant PrfA protein is set forth in SEQ ID NO: 82 (by SEQ ID NO: : Nucleic acid coding as described in 83). The complemented PrfA protein can be a homologue, variant, isoform, analog, fragment, homolog fragment, variant fragment, analog fragment or isoform fragment of SEQ ID NO: 80 or 82. PrfA protein can also be any otherListeria PrfA protein. Alternatively, the PrfA protein can be any other Gram-positive PrfA protein or any other PrfA protein. [0155] In another example, the bacterial strain or the Listeria strain may compriseactA Missing gene oractA An inactivating mutation in the gene, and the complement gene may comprise an actA gene to complement the mutation and restore the function of the Listeria strain. Other auxotrophic strains and supplemental systems can also be used with the methods and compositions provided herein. C. Preparation and Storage of Bacterial or Listeria Strains [0157] Recombinant bacterial strains (eg, Listeria strains) have been subcultured by animal hosts as appropriate. Such a subdivision can maximize the efficacy of the Listeria strain as a vaccine carrier, stabilize the immunogenicity of the Listeria strain, stabilize the pathogenicity of the Listeria strain, and increase the Listeria The immunogenicity of the strain can increase the pathogenicity of the Listeria strain, remove the unstable strain of the Listeria strain or reduce the occupancy rate of the unstable strain of the Listeria strain. Methods for substituting a recombinant Listeria strain in an animal host are well known in the art and are described, for example, in US 2006/0233835, which is hereby incorporated by reference in its entirety for all purposes. [0158] Recombinant bacterial strains (eg, Listeria strains) can be stored in a frozen cell bank or in a lyophilized cell bank. Such a cell bank can be, for example, a master cell bank, a working cell bank, or a Good Manufacturing Practice (GMP) cell bank. Examples of "good manufacturing practices" include those defined by 21 CFR 210-211 of the United States Code of Federal Regulations. However, "good manufacturing practices" may also be defined by other standards used to manufacture clinical grade materials or for human consumption, such as standards in countries other than the United States. Such cell banks may be intended for use in the manufacture of clinical grade materials or may conform to management practices for human use. [0159] Recombinant bacterial strains (eg, Listeria strains) may also be from a vaccine batch, from a frozen feedstock or from a lyophilized feedstock. Such cell banks, frozen stocks or vaccine dosages may exhibit greater than 90% viability, for example, after thawing. Thawing can be performed after cryopreservation storage or frozen storage for 24 hours. Alternatively, the storage may last for example 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 5 months, 6 months, 9 months or 1 year. [0161] The cell bank, frozen material or vaccine batch can be cryopreserved, for example, by growing a culture of a bacterial strain (eg, a Listeria strain) in a nutrient medium, and freezing in a solution containing glycerin. The culture, and storing the Listeria strain at below -20 °C. The temperature can be, for example, about -70 ° C or between about -70 ° C to about -80 ° C. Alternatively, the cell bank, frozen material or vaccine batch can be cryopreserved, for example, by growing a Listeria strain culture in a defined medium, and freezing the culture in a solution comprising glycerol, And storing the Listeria strain at below -20 °C. The temperature can be, for example, about -70 ° C or between about -70 ° C and about -80 ° C. Any component-determined microbial culture medium can be used in this method. Cultures (eg, cultures of Listeria vaccine strains used to produce Listeria vaccine dosages) can be derived, for example, from cell banks, self-frozen materials, starter cultures, or self-communities Vaccination. Cultures can be inoculated, for example, in the mid-log phase, mid-logarithmic growth phase, or another growth phase. In place of or in addition to glycerol, the solution for freezing optionally contains another additive or an additive having anti-freezing properties. Examples of such additives include, for example, mannitol, DMSO, sucrose, or any other number of additives or additives having anti-freezing properties. The nutrient medium for growing a culture of a bacterial strain (eg, a Listeria strain) can be any suitable nutrient medium. Examples of suitable media include, for example, LB; TB; a modified animal-free product, Terrific Broth; or a defined medium. [0165] The growth step can be carried out by any known means for growing bacteria. For example, the growth step can be performed with a shake flask (such as a shake flask with a baffle), a batch decimator, a stirred tank or flask, an airlift decimator, a fed-batch, a continuous cell reactor, and an immobilization. A cell reactor or any other means of growing bacteria is performed. [0166] Maintain a constant pH during culture growth (eg, in a batch fermentation), as appropriate. For example, the pH can be maintained at about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. Likewise, the pH can be, for example, from about 6.5 to about 7.5, from about 6.0 to about 8.0, from about 6.0 to about 7.0, from about 6.0 to about 7.0, or from about 6.5 to about 7.5. [0167] Optionally, a constant temperature can be maintained during growth of the culture. For example, the temperature can be maintained at about 37 ° C or at 37 ° C. Alternatively, the temperature can be maintained at 25 ° C, 27 ° C, 28 ° C, 30 ° C, 32 ° C, 34 ° C, 35 ° C, 36 ° C, 38 ° C or 39 ° C. [0168] Optionally, a constant dissolved oxygen concentration can be maintained during growth of the culture. For example, the dissolved oxygen concentration can be maintained at 20% saturation, 15% saturation, 16% saturation, 18% saturation, 22% saturation, 25% saturation, 30% saturation, 35% saturation, 40% saturation, 45% saturation, 50% saturated, 55% saturated, 60% saturated, 65% saturated, 70% saturated, 75% saturated, 80% saturated, 85% saturated, 90% saturated, 95% saturated, 100% saturated or nearly 100% saturated. [0169] Methods for lyophilizing and cryopreserving recombinant bacterial strains, such as Listeria strains, are known. For example, Listeria cultures can be rapidly frozen in liquid nitrogen followed by final freezing temperatures. Storage. Alternatively, the culture can be frozen in a more gradual manner (eg, by placing it in a culture vial at the final storage temperature). The culture can also be by any other method known to freeze bacterial cultures. [0170] The storage temperature of the culture can be, for example, between -20 ° C and -80 ° C. For example, the temperature can be significantly lower than -20 ° C or not higher than -70 ° C. Alternatively, the temperature can be about -70 ° C, -20 ° C, -30 ° C, -40 ° C, -50 ° C, -60 ° C, -80 ° C, -30 ° C to -70 ° C, -40 ° C to -70 ° C, -50 ° C to -70 °C, -60 ° C to -70 ° C, -30 ° C to -80 ° C, -40 ° C to -80 ° C, -50 ° C to -80 ° C, -60 ° C to -80 ° C or -70 ° C to -80 ° C. Alternatively, the temperature may be lower than -70 ° C or lower than -80 ° C. IV. Immunogenic composition, pharmaceutical composition and vaccine [0171] An immunogenic composition, a pharmaceutical composition or a vaccine is also provided, which comprises A recombinant fusion polypeptide disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide as disclosed herein, or a recombinant bacterium or Listeria strain as disclosed herein comprising an immunogenic composition of a Listeria strain may comprise Liss The strain of the genus is inherently immunogenic and/or the composition may further comprise an adjuvant. Other immunogenic compositions comprise a DNA immunotherapy or a peptide immunotherapeutic composition. [0172] The term "immunogenic composition" Means any composition comprising an antigen that, upon exposure to the composition, elicits an immune response against the antigen in the individual. The immune response elicited by the immunogenic composition can be directed against or against the particular antigen Specific epitopes. The immunogenic composition may comprise a single recombinant fusion polypeptide as disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide as disclosed herein, or a recombinant bacterium or Listeria as disclosed herein. A strain, or it may comprise a plurality of different recombinant fusion polypeptides as disclosed herein, encoding as disclosed herein A nucleic acid comprising a fusion polypeptide or a recombinant bacterial or Listeria strain as disclosed herein. For example, if the first recombinant fusion polypeptide comprises an antigenic peptide not comprising the second recombinant fusion polypeptide, the first recombinant fusion polypeptide is The second recombinant fusion polypeptide is different. The two recombinant fusion polypeptides may comprise some of the same antigenic peptides and are still considered different. Such different recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides or recombinant bacteria or Listeria strains may be Simultaneous administration to an individual or sequentially to an individual. When a drug substance comprising a recombinant Listeria strain (or recombinant fusion polypeptide or nucleic acid) disclosed herein is in a different dosage form (eg, one agent is a lozenge or capsule and the other One agent is a sterile liquid) and/or administered in different dosing schedules (eg, one composition from the mixture is administered at least daily and the other is administered less frequently, such as once a week, once every two weeks, or Sequential administration can be especially useful whenever it is done every three weeks. Multiple recombinant fusion polypeptides\nucleic acids encoding recombinant fusion polypeptides or recombinant bacteria or Listeria strains may each comprise a different antigenic peptide group. Alternatively, the recombinant fusion polypeptide \ nucleic acid encoding the recombinant fusion polypeptide or both or more than the recombinant bacterial or Listeria strain may comprise the same antigenic peptide group (eg, comprising the same set of antigenic peptides in a different order). The immunogenic composition may additionally comprise an adjuvant (eg, two or more than two adjuvants), a cytokine, a chemokine, or a combination thereof. Optionally, the immunogenic composition may additionally comprise an antigen presenting cell (APC), which may be autologous or may be allogeneic to the individual. The term adjuvant includes a compound or mixture that enhances an immune response against an antigen. For example, an adjuvant can be a non-specific stimulating agent for an immune response or a substance that allows for the production of a stock in an individual, the adjuvant providing even greater enhancement when combined with the immunogenic compositions disclosed herein and/or Long-term immune response. Adjuvants can promote, for example, a major Th1-mediated immune response, a Th1-type immune response, or a Th1-mediated immune response. Likewise, adjuvants may facilitate cell-mediated immune responses relative to antibody-mediated responses. Alternatively, the adjuvant may facilitate antibody mediated reactions. Some adjuvants can enhance the immune response by slowly releasing the antigen, while other adjuvants can mediate their effects by any of the following mechanisms: increasing cell infiltration, inflammation, and migration to the site of injection, specifically for antigens Presenting cells (APC); promoting the activation state of APC by up-regulating co-stimulatory signals or major histocompatibility complex (MHC) expression; enhancing antigen presentation; or inducing cytokine release for indirect effects. Examples of adjuvants include saponin QS21, CpG oligonucleotides, oligonucleotides containing unmethylated CpG, MPL, TLR agonists, TLR4 agonists, TLR9 agonists, Resiquimod^® , imiquimod, cytokines or nucleic acids encoding such cytokines, chemokines or nucleic acids encoding such chemokines, IL-12 or nucleic acids encoding IL-12, IL-6 or encoding IL -6 nucleic acids and lipopolysaccharides. Another example of a suitable adjuvant is Montanide ISA 51. Montanide ISA 51 contains natural metabolisable oils and refined emulsifiers. Yet another example of a suitable adjuvant is the detoxified Listeria lysin O (dtLLO) protein. An example of a dtLLO suitable for use as an adjuvant is encoded by SEQ ID NO:115. dtLLO encoded by a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 115 is also suitable for use as an adjuvant. Other examples of suitable adjuvants include granulocyte/macrophage colony-stimulating factor (GM-CSF) or nucleic acid encoding GM-CSF and keyhole limpet hemocyanin (KLH) a protein or a nucleic acid encoding the same. GM-CSF can be, for example, in yeast (Saccharomyces cerevisiae (S. cerevisiae )) Human proteins grown in the vector. GM-CSF promotes the asexual expansion and differentiation of hematopoietic progenitor cells, antigen presenting cells (APCs), dendritic cells and T cells. Still other examples of adjuvants include growth factors or nucleic acids encoding such growth factors, cell populations, Freund's incomplete adjuvant, aluminum phosphate, aluminum hydroxide, bacille Calmette-Guerin (BCG), alum, interleukin Or a nucleic acid encoding such interleukins, quill glycoside, monophosphoryl lipid A, liposomes, bacterial mitogens, bacterial toxins or any other type of known adjuvant (see, for example, Fundamental Immunology, 5th Edition ( August 2003): William E. Paul (editor); Lippincott Williams & Wilkins Publishers; Chapter 43: Vaccines, GJV Nossal, which is hereby incorporated by reference in its entirety for all purposes. The immunogenic composition may further comprise one or more immunomodulatory molecules. Examples include interferon gamma, cytokines, chemokines, and T cell stimulators. The immunogenic composition can be in the form of a vaccine or a pharmaceutical composition. The terms "vaccine" and "pharmaceutical composition" are interchangeable and refer to a pharmaceutically acceptable carrier containing an immunogenic composition for administration to an individual in vivo. The vaccine can be, for example, a peptide vaccine (eg, comprising a recombinant fusion polypeptide as disclosed herein), a DNA vaccine (eg, comprising a nucleic acid encoding a recombinant fusion polypeptide as disclosed herein), or a vaccine contained within a cell and delivered by a cell ( For example, recombination as disclosed hereinListeria ). The vaccine may prevent the individual from becoming infected or suffering from a disease or condition, and/or the vaccine may be therapeutic for an individual having the disease or condition. Methods for preparing peptide vaccines are well known and are described, for example, in EP 1408048, US 2007/0154953 and Ogasawara et al. (1992).Proc. Natl Acad Sci USA 89:8995-8999, each of which is incorporated herein by reference in its entirety for all purposes. Peptide evolution techniques can be used to generate antigens with higher immunogenicity, as appropriate. Techniques for the evolution of peptides are well known and are described, for example, in US 6,773,900, which is hereby incorporated by reference in its entirety for all purposes. "Pharmaceutically acceptable carrier" means a vehicle for containing an immunogenic composition which can be introduced into an individual without significant adverse effects and having no adverse effects on the immunogenic composition. . That is, "pharmaceutically acceptable" means that any formulation is safe and suitably delivers an effective amount of at least one immunogenic composition for use in the methods disclosed herein to the desired route of administration. Pharmaceutically acceptable carriers or vehicles or excipients are well known. Descriptions of suitable pharmaceutically acceptable carriers and factors relating to their selection are found in a variety of readily available sources, such asRemington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference in its entirety for all purposes. Such carriers can be adapted for any route of administration (eg, parenteral, enteral (eg, oral) or topical). Such pharmaceutical compositions can be buffered, for example, wherein the pH is maintained at a specific value ranging from pH 4.0 to pH 9.0 depending on the stability of the immunogenic composition and the route of administration. Suitable pharmaceutically acceptable carriers include, for example, sterile water; saline solutions such as physiological saline; dextrose; buffer solutions such as phosphate buffer or bicarbonate buffer solutions; alcohols; gum arabic; vegetable oils; Benzyl alcohol; polyethylene glycol; gelatin; carbohydrates (eg lactose, amylose or starch); magnesium stearate; talc; tannic acid; viscous paraffin; white paraffin; glycerol; alginate; hyaluronic acid; ; fragrance oil; fatty acid monoglyceride and diglyceride; pentaerythritol fatty acid ester; hydroxymethyl cellulose; polyvinylpyrrolidone and the like. Pharmaceutical compositions or vaccines may also include adjuvants that do not deleteriously react with the immunogenic composition, including, for example, diluents, stabilizers (eg, sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffers. Agents, viscosity enhancing additives, lubricants, salts that affect osmotic pressure, buffers, vitamins, colorants, flavoring agents, aromatic substances, and the like. For example, for liquid formulations, the pharmaceutically acceptable carrier can be an aqueous or non-aqueous solution, suspension, emulsion or oil. Non-aqueous solvents include, for example, propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic/aqueous solutions, emulsions or suspensions, including physiological saline and buffering media. Examples of oils include oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil and cod liver oil. Solid carriers/diluents include, for example, gums, starches (eg, corn starch, pregelatinized starch), sugars (eg, lactose, mannitol, sucrose, dextrose), cellulosic materials (eg, microcrystalline fibers) An acrylate (for example, polymethacrylate), calcium carbonate, magnesium oxide, talc or a mixture thereof. [0182] A pharmaceutical composition or vaccine that is continuously or directionally released may be provided as appropriate. This can be achieved, for example, via the use of liposomes or compositions in which the active compounds are protected by different decomposable coatings (e.g., by microencapsulation, multi-layer coating, etc.). Such compositions can be formulated for immediate or slow release. It is also possible to freeze-dry the composition and use the obtained lyophilizate (for example, for preparing an injectable product). The immunogenic compositions, pharmaceutical compositions or vaccines disclosed herein may also comprise one or more additional compounds effective to prevent or treat cancer. For example, additional compounds may comprise compounds suitable for use in chemotherapy, such as amsacrine, bleomycin, busulfan, capecitabine, carboplatin ), carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cristantaspase, ring Phosphonamine, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, table Epirubicin, etoposide, fludarabine, fluorouracil (5-FU), gemcitabine, gliadel implant, hydroxyurea, idarubicin, Isocyclophosphamide, irinotecan, leucovorin, liposomal cranberry, liposomal daunorubicin, lomustine, melphalan,巯基嘌呤, mesna, methotrexate, mitogen (mitomycin), mitoxantrone, oxaliplatin, paclitaxel (Taxol), pemetrexed, pentostatin, procarbazine , raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa ), thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine or combination. Additional compounds may also contain other biological agents, including Herceptin for the HER2 antigen.^® (trastuzumab), Avastin for VEGF^® (bevacizumab) or an antibody against the EGF receptor, such as Erbitux^® (cetuximab), duvalumumab (Medi4736) and Vectibix^® (panitumumab). Additional compounds may also include, for example, additional immunotherapy. Additional compounds may also comprise immunological checkpoint inhibitor antagonists, such as PD-1 signaling pathway inhibitors, CD-80/86 and CTLA-4 signaling pathway inhibitors, T cell membrane protein 3 (TIM3) signaling Pathway inhibitor, adenosine A2A receptor (A2aR) signaling pathway inhibitor, lymphocyte activation gene 3 (LAG3) signaling pathway inhibitor, killer immunoglobulin receptor (KIR) signaling pathway inhibitor, CD40 signaling Pathway inhibitor or any other antigen presenting cell/T cell signaling pathway inhibitor. Examples of immunological checkpoint inhibitor antagonists include anti-PD-L1/PD-L2 antibodies or fragments thereof, anti-PD-1 antibodies or fragments thereof, anti-CTLA-4 antibodies or fragments thereof or anti-B7-H4 antibodies or fragments thereof. An example of an anti-PD-1 antibody is Opdivo (nivolumab), an anti-PD-1 monoclonal antibody. In a specific embodiment, a combination therapy comprising a recombinant Listeria strain and Opdivo (Navuximab) is used to treat an individual having metastatic cervical cancer, the recombinant Listeria strain comprising a coding fusion polypeptide comprising The first open reading frame nucleic acid, wherein the fusion polypeptide comprises a PEST-containing peptide fused to an HPV16 antigen peptide and an HPV18 antigen peptide, wherein the HPV16 antigen peptide and the HPV18 antigen peptide are operably linked in series, wherein the HPV16 antigen peptide It is an HPV16 E6 antigen peptide or an HPV16 E7 antigen peptide, and wherein the HPV18 antigen peptide is an HPV18 E6 antigen peptide or an HPV18 E7 antigen peptide. Advaxis immunotherapy (ADVANCE) study using navumab to treat recurrent or metastatic cervical cancer is the development of second-line treatment for cervical cancer in first-line treatment failure in women with recurrent or metastatic cervical cancer. The ADVANCE study was a randomized phase global study in which more than 500 patients were randomized to patients with recurrent or metastatic cervical cancer who failed first-line therapy or were eligible for first-line therapy, compared to the single-agent chemistry selected by the investigator. Therapy evaluates the safety and efficacy of ADXS-602 (ADXS-DUAL) in combination with Navumab. Additional compounds may also comprise T cell stimulating agents, such as antibodies that bind to T cell receptor costimulatory molecules, antigen presenting cell receptors that bind to costimulatory molecules, or members of the TNF receptor superfamily, or their functionality. Fragment. The T cell receptor costimulatory molecule can comprise, for example, CD28 or ICOS. An antigen presenting cell receptor that binds to a costimulatory molecule can comprise, for example, a CD80 receptor, a CD86 receptor, or a CD46 receptor. Members of the TNF receptor superfamily may comprise, for example, a glucocorticoid-induced TNF receptor (GITR), OX40 (CD134 receptor), 4-1BB (CD137 receptor) or TNFR25.See for example WO2016100929, WO2016011362 and WO2016011357, each of which is incorporated by reference in its entirety for all purposes. V. Methods of Treatment [0187] The recombinant fusion polypeptides disclosed herein, nucleic acids encoding recombinant fusion polypeptides, recombinant bacterial or Listeria strains, immunogenic compositions, pharmaceutical compositions, and vaccines can be used in a variety of methods. For example, it can be used in a method of inducing an immune response against a tumor-associated antigen in an individual, in a method of inducing an anti-tumor or anti-cancer immune response in an individual, in a method of treating a tumor or cancer in an individual, in an individual A method of preventing a tumor or cancer or a method of protecting an individual from a tumor or cancer. It can also be used in a method for increasing the ratio of T effector cells to regulatory T cells (Tregs) in an individual's spleen and tumor, wherein the T effector cells target tumor associated antigens. It can also be used to increase tumor-associated antigen T cells in an individual, increase the survival time of an individual having a tumor or cancer, delay the onset of cancer in an individual, or reduce the size of a tumor or cancer metastasis in an individual. The tumor or cancer in any of the above methods may be, for example, an HPV-related cancer, such as a cervical tumor or cancer, an anal tumor or cancer, a head and neck tumor or cancer, or an oropharyngeal tumor or cancer. The cancer in any of the methods described herein can be metastatic cervical cancer. A method of inducing an anti-HPV16 and/or anti-HPV18 immune response in an individual can comprise, for example, administering to the individual a recombinant fusion polypeptide disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacterium or a Listeria strain An immunogenic composition, pharmaceutical composition or vaccine (eg, comprising a recombinant fusion polypeptide comprising a HPV16 and HPV18 antigenic peptide or a nucleic acid encoding the recombinant fusion polypeptide). Thereby, an anti-HPV16 and/or anti-HPV18 immune response can be induced in the individual. For example, in the case of a recombinant Listeria strain, the Listeria strain can express a fusion polypeptide thereby eliciting an immune response in the individual. The immune response can comprise, for example, a T cell response, such as a CD4+FoxP3-T cell response, a CD8+ T cell response, or a CD4+FoxP3- and CD8+ T cell response. Such methods can also increase the ratio of T effector cells to regulatory T cells (Tregs) in an individual's spleen and tumor microenvironment, thereby allowing for a deeper anti-tumor response in an individual. A method of inducing an anti-tumor or anti-cancer immune response in an individual can comprise, for example, administering to the individual a recombinant fusion polypeptide disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacterium or a Listeria strain, immunizing An original composition, a pharmaceutical composition or a vaccine. Thereby an anti-tumor or anti-cancer immune response can be induced in the individual. For example, in the case of a recombinant Listeria strain, the Listeria strain can express a fusion polypeptide thereby eliciting an anti-tumor or anti-cancer response in the individual. A method of treating a tumor or cancer in an individual (eg, wherein the tumor or cancer expresses HPV 16 and/or HPV 18) can comprise, for example, administering to the individual a recombinant fusion polypeptide disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide. , a recombinant bacterial or Listeria strain, an immunogenic composition, a pharmaceutical composition or a vaccine. The individual can then establish an immune response against a tumor or cancer that exhibits HPV 16 and/or HPV 18 antigenic peptides, thereby treating the tumor or cancer in the individual. A method of preventing a tumor or cancer in an individual or protecting the individual from a tumor or cancer (eg, wherein the tumor or cancer is associated with the performance of HPV 16 and/or HPV 18) can comprise, for example, administering to the individual Recombinant fusion polypeptides disclosed, nucleic acids encoding recombinant fusion polypeptides, recombinant bacterial or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines. The individual can then establish an immune response against the HPV 16 and/or HPV 18 antigenic peptide, thereby preventing the tumor or cancer or protecting the individual from tumor or cancer. In some of the above methods, administering two or more recombinant fusion polypeptides, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacterial or Listeria strain, an immunogenic composition, a pharmaceutical composition or a vaccine . The plurality of recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacterial or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines can be administered simultaneously in any order or combination or in any combination. As an example, if four different Listeria strains are administered, they may be administered sequentially, they may be administered simultaneously, or they may be administered in any combination (eg, simultaneously administering the first and second strains and Subsequently, the third and fourth strains were simultaneously administered). Optionally, in the case of sequential administration, the compositions may be administered during the same immune response, preferably within 0-10 days or 3-7 days of each other. A plurality of recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacterial or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines may each comprise a different antigenic peptide group. Alternatively, two or more than two may comprise the same set of antigenic peptides (eg, comprising the same set of antigenic peptides in a different order). Cancer is a physiological condition in a mammal that is typically characterized by the growth and proliferation of unregulated cells. HPV-related cancers (eg, cancers caused by HPV) include, for example, vaginal, vulvar penis, anus, rectum, and other cancers. For example, HPV-related cancers include, but are not limited to, cervical cancer, anal cancer, head and neck cancer, and oropharyngeal cancer. In general, HPV is thought to cause more than 90% of anal cancer and cervical cancer and more than 50% of vaginal, vulvar and penile cancer. Head and neck cancers are mostly caused by tobacco and alcohol, but recent studies have shown that about 60% to 70% of oropharyngeal cancers may be associated with HPV. The term "treat/treating" refers to both therapeutic and prophylactic or preventative measures, wherein the goal is to prevent or alleviate the target tumor or cancer. Examples of target tumors or cancers include, but are not limited to, cervical tumors or cancers, anal tumors or cancers, head and neck tumors or cancers or oropharyngeal tumors or cancers. Treatment may include one or more of the following: directly affecting or curing, curbing, inhibiting, preventing, or preventing a tumor, cancer, reducing its severity, delaying its onset, slowing its progression, stabilizing its progression, inducing its relief, prevention, or delay It has cancer metastasis, reduces/improves symptoms associated with it, or a combination thereof. For example, treatment can include increasing the expected survival time or reducing the size of the tumor or cancer metastasis. Effects (eg, containment, inhibition, prevention, reduction in severity, delayed onset, slowing progression, stabilizing progression, inducing remission, preventing or delaying cancer metastasis, reducing/ameliorating symptoms, etc. may be relative to untreated or receiving placebo Controlled individual for treatment. The term "treat/treating" can also refer to increasing the percentage of survival in an individual with a tumor or cancer or increasing the expected survival time (eg, relative to a treatment that is not treated or treated with placebo) Individual) In one example, "treatment" refers to delaying progression, accelerating remission, inducing remission, enhancing remission, accelerating recovery, increasing the efficacy of an alternative therapeutic, reducing resistance to an alternative therapeutic, or a combination thereof (eg, Relative to untreated or placebo-treated control individuals. The term "preventing" or "preventing" may refer to, for example, delaying the onset of symptoms, preventing tumor or cancer recurrence, reducing the number or frequency of recurrent events, and increasing symptomatic events. Latent time, cancer metastasis to prevent cancer or cancer, or a combination thereof. The term "containment" or "inhibition" may refer to For example, reducing the severity of symptoms, reducing the severity of acute events, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, improving symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof. The term "individual" refers to a mammal (eg, a human) that is required for the treatment of a tumor or cancer or that is susceptible to a tumor or cancer. The term individual also refers to a mammal (eg, a human) that is being treated prophylactically or therapeutically. Individuals may include dogs, cats, pigs, cows, sheep, goats, horses, rats, mice, non-human mammals, and humans. The term "individual" does not necessarily exclude health in all respects and does not have or exhibit cancer. Individuals with signs or tumors. [0196] If an individual has at least one known risk factor (eg, genetic, biochemical, family history, and contextual exposure), the risk of developing a tumor or cancer in an individual with the risk factor is statistical. If the individual is significantly larger than the individual who does not have the risk factor, the individual is at increased risk of developing a tumor or cancer. 197] "Symptoms" or "signs" means objective evidence of a disease as observed by a physician or subjective evidence of a disease as perceived by an individual, such as a change in gait. Symptoms or signs may be any manifestation of the disease. Symptoms may be Hair or secondary. The term "primary" refers to the direct result of a symptom or a specific disease (such as a tumor or cancer), and the term "secondary" refers to a symptom that is caused by or caused by the cause of the original. The disclosed recombinant fusion polypeptides, nucleic acids encoding the recombinant fusion polypeptides, immunogenic compositions, pharmaceutical compositions, and vaccines can treat primary or secondary symptoms or secondary complications. [0198] Recombination in an effective regimen A fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacterium or a Listeria strain, an immunogenic composition, a pharmaceutical composition or a vaccine, the effective scheme means the following dosage, administration route and frequency of administration, which delays tumor or cancer Attack, reduce its severity, inhibit its further deterioration, and/or improve at least one of its signs or symptoms. Alternatively, the recombinant fusion polypeptide, the nucleic acid encoding the recombinant fusion polypeptide, the recombinant bacterial or Listeria strain, the immunogenic composition, the pharmaceutical composition or the vaccine are administered in an effective protocol, and the effective solution means the following dosage and administration Route and frequency of administration that induces an immune response against a recombinant fusion polypeptide (or encoded by a nucleic acid), a recombinant or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a heterologous antigen in a vaccine, or in a recombination In the case of a bacterial or Listeria strain, an immune response against the bacterial or Listeria strain itself is induced. If the individual has developed a tumor or cancer, the regimen can be referred to as a therapeutically effective regimen. If an individual has an increased risk of developing a tumor or cancer relative to the general population, but has not experienced symptoms, the regimen can be referred to as a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy may be observed in individual patients relative to historical controls or past experiences of the same patient. In other instances, therapeutic or prophylactic efficacy may be demonstrated in a pre-clinical or clinical trial in a population of treated patients relative to a control population of untreated patients. For example, if the results achieved by an individual treated patient are more favorable than the average result in a control population of a similar patient not treated by the methods described herein, or if in a controlled clinical trial (eg, Phase II, II/) In phase III or phase III trials, a more favorable result is demonstrated in the treated patient versus the control patient at p < 0.05 or 0.01 or even 0.001 level, and the regimen can be considered therapeutically or prophylactically effective. An exemplary dose of a recombinant Listeria strain is, for example, 1 × 10⁶ - 1 × 10⁷ CFU, 1 × 10⁷ - 1 × 10⁸ CFU, 1 × 10⁸ - 3.31 × 10¹⁰ CFU, 1 × 10⁹ - 3.31 × 10¹⁰ CFU, 5-500 × 10⁸ CFU, 7-500 × 10⁸ CFU, 10-500 × 10⁸ CFU, 20-500 × 10⁸ CFU, 30-500 × 10⁸ CFU, 50-500 × 10⁸ CFU, 70-500 × 10⁸ CFU, 100-500 × 10⁸ CFU, 150-500 × 10⁸ CFU, 5-300 × 10⁸ CFU, 5-200 × 10⁸ CFU, 5-15 × 10⁸ CFU, 5-100 × 10⁸ CFU, 5-70 × 10⁸ CFU, 5-50 × 10⁸ CFU, 5-30 × 10⁸ CFU, 5-20 × 10⁸ CFU, 1-30 × 10⁹ CFU, 1-20 × 10⁹ CFU, 2-30 × 10⁹ CFU, 1-10 × 10⁹ CFU, 2-10 × 10⁹ CFU, 3-10 × 10⁹ CFU, 2-7 × 10⁹ CFU, 2-5 × 10⁹ CFU and 3-5 × 10⁹ CFU. Other exemplary doses of recombinant Listeria strains are, for example, 1 x 10⁷ Organs, 1.5 × 10⁷ Organs, 2 × 10⁸ Organs, 3 × 10⁷ Organs, 4 × 10⁷ Organs, 5 × 10⁷ Organs, 6 × 10⁷ Organs, 7 × 10⁷ Organs, 8 × 10⁷ Organs, 10 × 10⁷ Organs, 1.5 × 10⁸ Organs, 2 × 10⁸ Organs, 2.5 × 10⁸ Organs, 3 × 10⁸ Organs, 3.3 × 10⁸ Organs, 4 × 10⁸ Organs, 5 × 10⁸ Organs, 1 × 10⁹ Organs, 1.5 × 10⁹ Organs, 2 × 10⁹ Organs, 3 × 10⁹ Organs, 4 × 10⁹ Organs, 5 × 10⁹ Organs, 6 × 10⁹ Organs, 7 × 10⁹ Organs, 8 × 10⁹ Organs, 10 × 10⁹ Organs, 1.5 × 10¹⁰ Organs, 2 × 10¹⁰ Organs, 2.5 × 10¹⁰ Organs, 3 × 10¹⁰ Organs, 3.3 × 10¹⁰ Organs, 4 × 10¹⁰ Organs and 5 × 10¹⁰ An organism. The dosage may depend on the condition of the patient and the response to prior treatment (if present, whether the treatment is prophylactic or therapeutic) and other factors. [0200] Administration can be by any suitable means. For example, the administration may be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraventricular, intraperitoneal, topical, intranasal, intramuscular, intraocular, rectal, conjunctiva, Percutaneous, intradermal, transvaginal, transrectal, intratumoral, paracancerous, transmucosal, intravascular, intraventricular, inhalation (aerosol), nasal aspiration (spray), sublingual, aerosol, suppository Or a combination thereof. For intranasal administration or administration by inhalation, a recombinant fusion polypeptide which is mixed and aerosolized or atomized in the presence of a suitable carrier, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacterial or Listeria strain, an immunogenic composition A solution or suspension of the pharmaceutical composition or vaccine is suitable. Such aerosols can comprise any of the recombinant fusion polypeptides described herein, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacterial or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine. Administration may also be in the form of a suppository (e.g., a rectal suppository or a urethral suppository), in the form of a pellet for subcutaneous implantation (e.g., controlled release over a period of time) or in the form of a capsule. Administration can also be by injection into a tumor site or injection into a tumor. The dosage regimen can be readily determined based on factors such as the precise nature and type of tumor or cancer to be treated, the severity of the tumor or cancer, the age and general physiological conditions of the individual, the weight of the individual, the response of the individual, and the like. factor. The frequency of administration may depend on the recombinant fusion polypeptide, the nucleic acid encoding the recombinant fusion polypeptide, the recombinant bacterium or the Listeria strain, the immunogenic composition, the pharmaceutical composition, or the half-life of the vaccine in the individual, the individual conditions and the route of administration. And other factors. The frequency can be, for example, daily, weekly, monthly, quarterly, or an irregular time interval in response to changes in individual conditions or progression of the tumor or cancer to be treated. The duration of treatment may depend on individual conditions and other factors. For example, the treatment schedule can be several weeks, several months, or several years (eg, up to 2 years). For example, repeated administration (administration) can be performed immediately following the first treatment schedule or after a time interval of days, weeks, or months to achieve tumor regression or tumor growth inhibition. The assessment can be determined by any known technique, including diagnostic methods such as imaging techniques, serum tumor marker analysis, biopsy, or the presence, absence or improvement of tumor associated symptoms. As a specific example, the recombinant fusion polypeptide, the nucleic acid encoding the recombinant fusion polypeptide, the recombinant bacterial or Listeria strain, the immunogenic composition, the pharmaceutical composition or the vaccine can be administered once every 3 weeks for up to 2 years. In one example, a recombinant fusion polypeptide disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacterial or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine is administered in increasing doses to increase T The ratio of effector cells to regulatory T cells and produces a more potent anti-tumor immune response. Anti-tumor immune responses can be further enhanced by providing the individual with cytokines including, for example, IFN-[gamma], TNF-[alpha], and other cytokines known to enhance cellular immune responses. See for example, US 6,991,785, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. Some methods may further comprise "adding" additional recombinant fusion polypeptides to the individual, nucleic acids encoding the recombinant fusion polypeptide, recombinant bacterial or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines, or multiple doses. A recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacterial or Listeria strain, an immunogenic composition, a pharmaceutical composition or a vaccine. "Additional" refers to an individual who is administered an additional dose. For example, in some methods, two additions (or a total of three vaccinations) are administered, three additions are made, four additions are made, five additions are made, or six or more additions are made. . The number of administrations administered may depend on, for example, the response of the tumor or cancer to the treatment. Depending on the case, the recombinant fusion polypeptide used in the booster vaccination, the nucleic acid encoding the recombinant fusion polypeptide, the recombinant bacterium or the Listeria strain, the immunogenic composition, the pharmaceutical composition or the vaccine is initially "initial ( Priming) The recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition or vaccine used in the vaccination are the same. Alternatively, the applicator recombinant fusion polypeptide, recombinant bacterial or Listeria strain, immunogenic composition, pharmaceutical composition or vaccine and initial recombinant fusion polypeptide, recombinant bacterial or Listeria strain, immunogenic composition, The pharmaceutical composition or vaccine is different. The same dose is used in the initial and additional vaccinations, as appropriate. Alternatively, a larger dose is used in the supplement or a smaller dose is used in the supplement. The period between the initial and additional vaccination can be determined experimentally. For example, the period between initial and additional vaccination can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6-8 weeks, or 8-10 weeks. [0204] A heterologous initial addition strategy has been effective in enhancing the immune response and providing protection against many pathogens. See, for example, Schneider et al. (1999)Immunol. Rev. 170:29-38; Robinson (2002)Nat. Rev. Immunol 2:239-250; Gonzalo et al. (2002)Vaccine 20:1226-1231; and Tanghe (2001)Infect. Immun 69:3041-3047, each of which is incorporated herein by reference in its entirety for all purposes. Providing antigens in different forms during initial and additional injections maximizes the immune response to the antigen. The DNA vaccine is initially added with a protein-containing adjuvant or the DNA encoding the antigen is added by a viral vector to add an antigen-specific antibody and CD4.⁺ T cell response or CD8⁺ An effective way of T cell response. See, for example, Shiver et al. (2002)Nature 415:331-335; Gilbert et al. (2002)Vaccine 20:1039-1045; Billaut-Mulot et al. (2000)Vaccine 19:95-102; and Sin et al. (1999)DNA Cell Biol. 18: 771-779, each of which is incorporated herein by reference in its entirety for all purposes. As an example, when an individual is initially vaccinated with DNA and then added with an antigen-expressing adenoviral vector, adding CRL1005 poloxamer (12 kDa, 5% POE) to the DNA encoding the antigen enhances T. Cellular response.See for example , Shiver et al. (2002)Nature 415:331-335, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. As another example, a vector construct encoding an immunogenic portion of an antigen and a protein comprising an immunogenic portion of the antigen can be administered.See for example, US 2002/0165172, which is hereby incorporated by reference in its entirety for all purposes. Similarly, the immune response to nucleic acid vaccination can be enhanced by simultaneously administering (eg, during the same immune response, preferably within 0-10 days or 3-7 days of each other) the polynucleotides and polypeptides of interest. .See for example US 6,500, 432, which is hereby incorporated by reference in its entirety for all purposes. The methods of treatment disclosed herein may also comprise administering one or more additional compounds effective to prevent or treat cancer. For example, additional compounds may comprise compounds suitable for use in chemotherapy, such as amsacrine, bleomycin, busulfan, capecitabine, carboplatin ), carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cristantaspase, ring Phosphonamine, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, table Epirubicin, etoposide, fludarabine, fluorouracil (5-FU), gemcitabine, gliadel implant, hydroxyurea, idarubicin, Isocyclophosphamide, irinotecan, leucovorin, liposomal cranberry, liposomal daunorubicin, lomustine, melphalan,巯基嘌呤, mesna, methotrexate, mitogen (mitomycin), mitoxantrone, oxaliplatin, paclitaxel (Taxol), pemetrexed, pentostatin, procarbazine , raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa ), thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine or combination. Alternatively, the additional compound may also comprise other biological agents, including Herceptin for the HER2 antigen.^® (trastuzumab), Avastin for VEGF^® (bevacizumab) or an antibody against the EGF receptor, such as Erbitux^® (cetuximab) and Vectibix^® (panitumumab). Alternatively, the additional compound may comprise other immunotherapies. Alternatively, the additional compound may be a guanamine 2,3-dioxygenase (IDO) pathway inhibitor such as 1-methyltryptophanic acid (1MT), 1-methyltryptophanic acid (1MT), necrosis Prime-1, pyridoxal isonicotinium guanidine, ebselen (Ebselen), 5-methylindole-3-carbaldehyde, CAY10581 (an anti-IDO antibody) or small molecule IDO inhibitor. IDO inhibition enhances the efficacy of chemotherapeutic agents. The methods of treatment disclosed herein can also be combined with radiation (eg, intensity modulated radiation therapy (IMRT)), stem cell therapy, surgery, or any other treatment. Such additional compounds or treatments may precede the administration of a recombinant fusion polypeptide disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacterial or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine, Following administration of a recombinant fusion polypeptide disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacterial or Listeria strain, an immunogenic composition, a pharmaceutical composition or vaccine, or a recombinant fusion polypeptide disclosed herein A nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacterial or Listeria strain, an immunogenic composition, a pharmaceutical composition or a vaccine. Targeted immunomodulatory therapies are primarily directed to the activation of costimulatory receptors, for example by using members of the tumor necrosis factor receptor superfamily (including 4-1BB, OX40, and GITR (glucocorticoid-induced TNF) The agonist antibody of the receptor-related)) is carried out. The possibility of modulating GITR in an anti-tumor and vaccine environment has been demonstrated. Another target of agonist antibodies is a costimulatory signaling molecule for T cell activation. Targeting costimulatory signaling molecules can result in enhanced T cell activation and a more potent immune response. Co-stimulation can also help prevent the inhibitory effects of checkpoint inhibition and increase antigen-specific T cell proliferation. Immunological treatment based on Listeria by inducing de novo inducing and destroying tumor-targeting tumor antigen-specific T cells and by reducing immunosuppressive regulatory T cells (Tregs) and bone marrow-derived suppressor cells in the tumor microenvironment The number and activity of (MDSC) come into play. Antibodies for T cell co-suppressive or costimulatory receptors (eg, checkpoint inhibitors CTLA-4, PD-1, TIM-3, LAG3, and co-stimulants CD137, OX40, GITR, and CD40) (or Functional fragments) may have synergistic effects with Listeria-based immunotherapy. Thus, some methods may comprise further administering a composition comprising an immunological checkpoint inhibitor antagonist, such as a PD-1 signaling pathway inhibitor, CD-80/86 and CTLA- 4 signaling pathway inhibitor, T cell membrane protein 3 (TIM3) signaling pathway inhibitor, adenosine A2A receptor (A2aR) signaling pathway inhibitor, lymphocyte activation gene 3 (LAG3) signaling pathway inhibitor, killer immunization A globin receptor (KIR) signaling pathway inhibitor, a CD40 signaling pathway inhibitor, or any other antigen presenting cell/T cell signaling pathway inhibitor. Examples of immunological checkpoint inhibitor antagonists include anti-PD-L1/PD-L2 antibodies or fragments thereof, anti-PD-1 antibodies or fragments thereof, anti-CTLA-4 antibodies or fragments thereof or anti-B7-H4 antibodies or fragments thereof. For example, anti-PD-1 antibodies can be 5-10 mg/kg every 2 weeks, 5-10 mg/kg every 3 weeks, 1-2 mg/kg every 3 weeks, 1-10 mg/kg per week, per Two weeks 1-10 mg/kg, 1-10 mg/kg every 3 weeks or 1-10 mg/kg every 4 weeks were administered to the individual. [0210] Likewise, some methods may further comprise administering a T cell stimulating agent, such as an antibody that binds to a T cell receptor costimulatory molecule, an antigen presenting cell receptor that binds to a costimulatory molecule, or a member of the TNF receptor superfamily. Or a functional fragment thereof. The T cell receptor costimulatory molecule can comprise, for example, CD28 or ICOS. An antigen presenting cell receptor that binds to a costimulatory molecule can comprise, for example, a CD80 receptor, a CD86 receptor, or a CD46 receptor. Members of the TNF receptor superfamily may comprise, for example, a glucocorticoid-induced TNF receptor (GITR), OX40 (CD134 receptor), 4-1BB (CD137 receptor) or TNFR25. For example, some methods can further comprise administering an effective amount of an antibody comprising a T cell receptor costimulatory molecule or a functional fragment thereof or binding to an antigen presenting cell receptor that binds to a costimulatory molecule. A composition of an antibody or a functional fragment thereof. The antibody may be, for example, an anti-TNF receptor antibody or antigen-binding fragment thereof (eg, a member of the TNF receptor superfamily, a glucocorticoid-induced TNF receptor (GITR), an OX40 (CD134 receptor), a 4-1BB (CD137 receptor) Or TNFR25), an anti-OX40 antibody or antigen-binding fragment thereof or an anti-GITR antibody or antigen-binding fragment thereof. Alternatively, other agonistic molecules can be administered (eg, GITRL, active fragments of GITRL, fusion proteins containing GITRL, fusion proteins containing active fragments of GITRL, antigen presenting cells (APC)/T cell agonists, CD134 Or a ligand or fragment thereof, CD137 or a ligand or fragment thereof, or an inducible T cell costimulator (ICOS) or a ligand or a fragment thereof, or a agonistic small molecule). In one embodiment, some methods can further comprise administering an anti-CTLA-4 antibody or a functional fragment thereof and/or an anti-CD137 antibody or a functional fragment thereof. For example, at about 72 hours or at the first time after the first administration of the recombinant fusion polypeptide, the nucleic acid encoding the recombinant fusion polypeptide, the recombinant bacterium or the Listeria strain, the immunogenic composition, the pharmaceutical composition or the vaccine Administration of anti-CTLA-4 antibody or functional fragment thereof about 48 hours after administration of the recombinant fusion polypeptide, nucleic acid encoding the recombinant fusion polypeptide, recombinant bacterial or Listeria strain, immunogenic composition, pharmaceutical composition or vaccine Or an anti-CD137 antibody or a functional fragment thereof. The anti-CTLA-4 antibody or a functional fragment thereof or an anti-CD137 antibody or a functional fragment thereof can be administered, for example, at a dose of about 0.05 mg/kg and about 5 mg/kg. The recombinant Listeria strain or the immunogenic composition comprising the recombinant Listeria strain may, for example, be about 1 x 10⁹ The dose of CFU is administered. Some such methods can further comprise administering an effective amount of an anti-PD-1 antibody or a functional fragment thereof. Methods for assessing the efficacy of cancer immunotherapy are well known and described, for example, in Dzojic et al. (2006)Prostate 66(8): 831-838; Naruishi et al. (2006)Cancer Gene Ther. 13(7): 658-663, Sehgal et al. (2006)Cancer Cell Int. 6:21) and Heinrich et al. (2007)Cancer Immunol Immunother 56(5): 725-730, each of which is incorporated herein by reference in its entirety for all purposes. As an example, for prostate cancer, a prostate cancer model can be tested for the methods and compositions disclosed herein, such as the TRAMP-C2 mouse model, the 178-2 BMA cell model, the PAIII adenocarcinoma model, the PC-3M model, or any Other prostate cancer models. Alternatively or additionally, immunotherapy can be tested in a human subject and the efficacy can be monitored using known individuals. Such methods can include, for example, direct measurement of CD4+ and CD8+ T cell responses, or measurement of disease progression (eg, by determining the number or size of tumor cancer metastases, or monitoring disease symptoms such as cough, chest pain, weight loss, etc. ). Methods for assessing the efficacy of cancer immunotherapy in human subjects are well known and described, for example, in Uenaka et al. (2007).Cancer Immun. 7:9 and Thomas-Kaskel et al. (2006)Int J Cancer Each of 119(10):2428-2434 is hereby incorporated by reference in its entirety for all purposes. VI. Kits [0215] Kits comprising reagents for performing the methods disclosed herein or kits comprising the compositions, tools or instruments disclosed herein are also provided. For example, such kits can comprise a recombinant fusion polypeptide disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide disclosed herein, a recombinant bacterium or Listeria strain disclosed herein, immunized as disclosed herein. The original composition, the pharmaceutical composition disclosed herein or the vaccine disclosed herein. Such kits may additionally comprise instructional materials describing the use of recombinant fusion polypeptides, nucleic acids encoding the recombinant fusion polypeptides, recombinant Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines for performing the methods disclosed herein . Such kits may further comprise an applicator as the case may be. Although model sets are described below, the contents of other applicable sets will become apparent in light of the present disclosure. [0217] All patent applications, websites, other publications, registration numbers, and the like, cited above or below, are hereby incorporated by reference in their entirety for all purposes in the extent of Instructions are incorporated by reference in their entirety. If the different versions of the different time series are related to the deposit number, it means the version associated with the deposit number at the effective filing date of this application. A valid filing date means the earlier of the application date of the actual application date or the priority application number referring to the deposit number, if applicable. Similarly, if a different version of the publication, website or the like is disclosed at different times, unless otherwise indicated, it means the most recently published version at the effective filing date of this application. Any feature, step, element, specific example or aspect of the invention may be used in any other combination, unless otherwise specifically indicated. Although the present invention has been described in considerable detail, by the claims List of Specific Examples [0218] The subject matter disclosed herein includes, but is not limited to, the following specific examples: 1. A recombinant Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion The polypeptide comprises a PEST-containing peptide fused to an HPV 16 antigen peptide and an HPV 18 antigen peptide, wherein the HPV 16 antigen peptide and the HPV 18 antigen peptide are operably linked in series. 2. The recombinant Listeria strain according to the specific example 1, wherein the HPV16 antigen peptide is an HPV16-E6 antigen peptide or an HPV16-E7 antigen peptide, and wherein the HPV18 antigen peptide is an HPV18-E6 antigen peptide or an HPV18-E7 antigen Peptide. 3. The recombinant Listeria strain of Specific Example 1, wherein the HPV 16 antigen peptide and the HPV 18 antigen peptide are directly fused to each other without an intervention sequence. 4. The recombinant Listeria strain of Specific Example 1, wherein the HPV-16 antigen peptide and the HPV-18 antigen peptide are linked to each other via a peptide linker. 5. The recombinant Listeria strain of specific example 4, wherein the peptide linker comprises one or more of the linkers set forth in SEQ ID NOs: 33-42. 6. The recombinant Listeria strain according to any one of the examples 1 to 5, wherein the HPV16 antigen peptide is an HPV16-E7 antigen peptide, and the HPV18 antigen peptide is an HPV18-E7 antigen peptide. 7. The recombinant Listeria strain of specific example 6, wherein the HPV16-E7 antigen peptide comprises at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of SEQ ID NO: A consensus sequence; and/or wherein the HPV 18-E7 antigen peptide comprises a sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:98. 8. The recombinant Listeria strain of specific example 7, wherein the segment encoding the open reading frame of the HPV 16-E7 antigen peptide comprises at least 90%, 95%, 96%, 97% of SEQ ID NO: 95, 98%, 99% or 100% identical sequence and encoding the peptide sequence set forth in SEQ ID NO: 96, and/or wherein the open reading frame encoding the HPV18-E7 antigenic peptide comprises SEQ ID NO : 97 at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical sequence and encodes the peptide sequence set forth in SEQ ID NO:98. 9. The recombinant Listeria strain of any one of embodiments 6 to 8, wherein the segment of the fusion polypeptide comprising the HPV 16 antigen peptide and the HPV 18 antigen peptide comprises at least 90 of any of the following sequences %, 95%, 96%, 97%, 98%, 99% or 100% identical sequences: SEQ ID NOs: 100, 102, 104, 106, 108, 110, 112 and 114. 10. The recombinant Listeria strain of the specific example 9, wherein the segment of the open reading frame encoding the segment of the fusion polypeptide comprising the HPV 16 antigen peptide and the HPV 18 antigen peptide comprises at least 90% of the following sequence, 95%, 96%, 97%, 98%, 99% or 100% identical sequence: SEQ ID NO: 99, 101, 103, 105, 107, 109, 111 or 113, and encoding the sequences set forth below, respectively : SEQ ID NO: 100, 102, 104, 106, 108, 110, 112 or 114. 11. The recombinant Listeria strain of any one of the specific examples 1 to 5, wherein the HPV16 antigen peptide is an HPV16-E6 antigen peptide, and the HPV18 antigen peptide is an HPV18-E6 antigen peptide. 12. The recombinant Listeria strain according to any one of the examples 1 to 5, wherein the HPV16 antigen peptide is an HPV16-E6 antigen peptide, and the HPV18 antigen peptide is an HPV18-E7 antigen peptide. 13. The recombinant Listeria strain according to any one of the examples 1 to 5, wherein the HPV16 antigen peptide is an HPV16-E7 antigen peptide, and the HPV18 antigen peptide is an HPV18-E6 antigen peptide. 14. The recombinant Listeria strain according to any of the preceding embodiments, wherein the fusion polypeptide further comprises one of the N-terminal and/or C-terminal of the HPV 16 antigen peptide and the HPV 18 antigen peptide operably linked in series. Or multiple peptide tags. 15. The recombinant Listeria strain of specific example 14, wherein the one or more peptide tags comprise one or more of the following: a 3 x FLAG tag; a 2 x FLAG tag, a 6 x His tag; and a SIINFEKL tag . 16. The recombinant Listeria strain according to the specific example 15, wherein the fusion polypeptide comprises a 3×FLAG tag at the N-terminus of the HPV16 antigen peptide operably linked in series and the HPV18 antigen peptide, and is at the antigen The SIINFEKL tag at the C-terminus of the peptide. 17. The recombinant Listeria strain of the specific example 15, wherein the fusion polypeptide comprises the SIINFEKL tag at the N-terminus of the HPV16 antigen peptide operably linked in series and the HPV18 antigen peptide, and at the antigenic peptide C-side 3 × FLAG tag. 18. The recombinant Listeria strain of specific example 15, wherein the fusion polypeptide comprises a 3xFLAG tag and a SIINFEKL tag at the C-terminus of the HPV16 antigen peptide operably linked in series and the HPV18 antigen peptide. 19. The recombinant Listeria strain of any of the preceding specific examples, wherein the PEST-containing peptide is at the N-terminus of the fusion polypeptide. 20. The recombinant Listeria strain according to any of the preceding specific examples, wherein the PEST-containing peptide is a Listeria lysin O (LLO) protein or a fragment thereof or an ActA protein or a fragment thereof. 21. The recombinant Listeria strain of specific example 20, wherein the Listeria lysin O (LLO) protein or a fragment thereof is an N-terminal fragment of LLO. 22. The recombinant Listeria strain of specific example 21, wherein the N-terminal fragment of the LLO has the sequence set forth in any one of SEQ ID NOs 57-59. 23. The recombinant Listeria strain of specific example 20, wherein the PEST-containing peptide is the LLO protein or a fragment thereof and comprises a mutation in a cholesterol binding domain. 24. The recombinant Listeria strain of specific example 23, wherein the LLO mutation comprises one of: (1) a substitution of residue C484, W491 or W492 of SEQ ID NO: 55 or when the LLO protein is SEQ ID NO: 55 the corresponding substitution at the optimal alignment; or (2) the deletion of 1-11 amino acids in residues 483-493 of SEQ ID NO: 55 or when the LLO protein and SEQ ID NO :55 The corresponding alignment is missing. 25. The recombinant Listeria strain of any of the preceding specific examples, wherein the nucleic acid is operably integrated into the Listeria genome. 26. The recombinant Listeria strain of any one of embodiments 1 to 24, wherein the nucleic acid is in an episomal form. 27. The recombinant Listeria strain of any of the preceding specific examples, wherein the nucleic acid does not confer antibiotic resistance to the recombinant Listeria strain. 28. The recombinant Listeria strain of any of the preceding specific examples, wherein the recombinant Listeria strain is attenuated. 29. The recombinant Listeria strain according to any of the preceding specific examples, wherein the recombinant Listeria strain is an auxotrophic Listeria strain. 30. The recombinant Listeria strain of the specific example 28 or 29, wherein the attenuated Listeria strain comprises a mutation in one or more endogenous genes that inactivates the one or more endogenous genes . 31. The recombinant Listeria strain of specific example 30, wherein the one or more endogenous genes compriseprfA . 32. The recombinant Listeria strain of specific example 30, wherein the one or more endogenous genes compriseactA . 33. The recombinant Listeria strain of specific example 30, wherein the one or more endogenous genes compriseactA andinlB . 34. The recombinant Listeria strain of specific example 30, wherein the one or more endogenous genes compriseactA ,Dal ,andDat . 35. The recombinant Listeria strain of any of the preceding specific examples, wherein the nucleic acid comprises a second open reading frame encoding a metabolic enzyme. 36. The recombinant Listeria strain of specific example 35, wherein the metabolic enzyme is a propylamine racemase or a D-amino acid transaminase. 37. The recombinant Listeria strain of any of the preceding specific examples, wherein the fusion polypeptide is self-Hly Promoter,prfA Promoter,actA Promoter orP60 Promoter performance. 38. The recombinant Listeria strain of the specific example 37, wherein the fusion polypeptide is self-Hly Promoter performance. 39. The recombinant Listeria strain according to any of the preceding specific examples, wherein the recombinant Listeria strain is a recombinant Listeria monocytogenes strain. 40. The recombinant Listeria strain of any one of the specific examples 1 to 19, wherein the recombinant Listeria strain is comprisedprfA Deletion or attenuation of inactivating mutations in prfAListeria monocytogenes A strain, wherein the nucleic acid is in an episomal plastid and comprises a second open reading frame encoding a D133V PrfA mutant protein. 41. The recombinant Listeria strain of any one of embodiments 1 to 19, wherein the recombinant Listeria strain is comprisedactA ,Dal andDat An attenuated Listeria monocytogenes strain having a deletion or an inactivating mutation in actA, dal, and dat, wherein the nucleic acid is in an episomal plastid and comprises a coding for alanine racemase or D-amino acid The second reading frame of the transaminase, and wherein the PEST-containing peptide is an N-terminal fragment of LLO. 42. The recombinant Listeria strain according to any one of embodiments 1 to 19, wherein the recombinant Listeria strain is comprisedactA andinlB The attenuated Listeria monocytogenes strain having a deletion or inactivation mutation, wherein the nucleic acid is genetically integrated, and wherein the PEST-containing peptide is an ActA protein or a fragment thereof. 43. The recombinant Listeria strain of any of the preceding specific examples, wherein the recombinant Listeria strain has been subcultured by an animal host. 44. The recombinant Listeria strain of any of the preceding specific examples, wherein the recombinant Listeria strain is capable of escaping phagosomes. 45. An immunogenic composition comprising a recombinant Listeria strain according to any of the preceding specific examples. 46. The immunogenic composition of embodiment 45, wherein the immunogenic composition further comprises an adjuvant. 47. The immunogenic composition of embodiment 46, wherein the adjuvant comprises a granule globule/macrophage colony stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, Monophosphoryl lipid A, dtLLO or an oligonucleotide containing unmethylated CpG. 48. A method of inducing an immune response against a tumor or cancer in an individual, comprising administering to the individual a recombinant Listeria strain of any one of Specific Examples 1 to 44 or any one of Specific Examples 45 to 47 An immunogenic composition. 49. A method of preventing or treating a tumor or cancer in an individual comprising immunizing the individual with a recombinant Listeria strain of any one of Examples 1 to 44 or any one of Specific Examples 45 to 47 Original composition. The method of any one of embodiments 48 to 49, wherein the method further comprises administering an immunological checkpoint inhibitor antagonist. 51. The method of embodiment 50, wherein the immunological checkpoint inhibitor comprises an anti-PD-1 antibody or antigen-binding fragment thereof and/or an anti-CTLA-4 antibody or antigen-binding fragment thereof. The method of any one of embodiments 48 to 51, wherein the method further comprises administering a T cell stimulating agent. 53. The method of embodiment 52, wherein the T cell stimulating agent comprises an anti-OX40 antibody or antigen-binding fragment thereof or an anti-GITR antibody or antigen-binding fragment thereof. The method of any one of embodiments 48 to 53, wherein the tumor or cancer is a cervical tumor or cancer, an anal tumor or cancer, a head and neck tumor or a cancer or an oropharyngeal tumor or cancer. 55. The method of embodiment 54, wherein the tumor or cancer is cancer metastasis. The method of any one of embodiments 48 to 55, wherein the tumor or cancer is HPV-16 positive. The method of any one of embodiments 48 to 55, wherein the tumor or cancer is HPV-18 positive. 58. A cell bank comprising a recombinant Listeria strain of one or more of any one of embodiments 1 to 44. 59. The cell bank of embodiment 58, wherein the cell bank is a frozen cell bank or a lyophilized cell bank. BRIEF DESCRIPTION OF THE SEQUENCES [0219] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using the standard letter abbreviations for nucleotide bases and the three-word code for amino acids. Nucleotide sequences follow a standard convention that begins at the 5' end of the sequence and proceeds in the forward direction (ie, from left to right in each line) to the 3' end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the presented strand. The amino acid sequence follows the standard convention that begins at the amino terminus of the sequence and proceeds in the forward direction (i.e., from left to right in each line) to the carboxy terminus. SIINFEKL tag DNA v.1 (SEQ ID NO: 1): GCACGTAGTATAATCAACTTTGAAAAACTGTAATAA DNA v.2 (SEQ ID NO: 2): GCACGTTCTATTATCAACTTCGAAAAACTATAATAA DNA v.3 (SEQ ID NO: 3): GCCCGCAGTATTATCAATTTCGAAAAATTATAATAA DNA v.4 (SEQ ID NO: 4): GCGCGCTCTATAATTAACTTCGAAAAACTTTAATAA DNA v.5 (SEQ ID NO: 5): GCACGCTCCATTATTAACTTTGAAAAACTTTAATAA DNA v.6 (SEQ ID NO: 6): GCTCGCTCTATCATCAATTTCGAAAAACTTTAATAA DNA v.7 (SEQ ID NO: 7): GCACGTAGTATTATTAACTTCGAAAAGTTATAATAA DNA v.8 (SEQ ID NO:8): GCACGTTCCATCATTAACTTTGAAAAACTATAATAA DNA v.9 (SEQ ID NO:9): GCTCGCTCAATCATCAACTTTGAAAAGCTATAATAA DNA v.10 (SEQ ID NO:10): GCTCGCTCTATCATCAACTTCGAAAAATTGTAATAA DNA v.11 (SEQ ID NO:11): GCTCGCTCTATTATCAATTTTGAAAAATTATAATAA DNA v.12 ( SEQ ID NO: 12): GCTCGTAGTATTATTAATTTCGAAAAATTATAATAA DNA v.13 (SEQ ID NO: 13): GCTCGTTCGATTATCAACTTCGAAAAACTGTAATAA DNA v.14 (SEQ ID NO: 14): GCAAGAAGCATCATCAACTTCGAAAAACTGTAATAA DNA v.15 (SEQ ID NO: 15): GCGCGTTCTATTATTAATTTTGAAAAATTATAATAA Protein (SEQ ID NO: 16): ARSIINFEKL 3×FLAG tag DNA v.1 (SEQ ID NO: 17): GATTATAAAGATCATGACGGAGACTATAAAGACCATGACATTGATTACAAAGACGACGATGACAAA DNA v.2 (SEQ ID NO: 18): GACTATAAAGACCACGATGGCGATTATAAAGACCATGATATTGACTACAAAGATGATGATGATAAG DNA v.3 (SEQ ID NO: 19): GATTATAAAGATCATGATGGCGACTATAAAGATCATGATATCGATTACAAAGATGACGATGACAAA DNA v.4 (SEQ ID NO: 20): GACTACAAAGATCACGATGGTGACTACAAAGATCACGACATTGATTATAAAGACGATGATGACAAA DNA v.5 (SEQ ID NO: 21): GATTACAAAGATCACGATGGTGATTATAAGGATCACGATATTGATTACAAAGACGACGACGATAAA DNA v.6 (SEQ ID NO: 22): GATTACAAAGATCACGATGGCGATTACAAAGATCATGACATTGACTACAAAGACGATGATGATAAA DNA v.7 (SEQ ID NO: 23) : GATTACAAGGATCATGATGGTGATTACAAAGATCACGATATCGACTACAAAGATGATGACGATAAA DNA v.8 (SEQ ID NO: 24): GACTACAAAGATCATGATGGTGATTACAAAGATCATGACATTGATTATAAAGATGATGATGACAAA DNA v.9 (SEQ ID NO: 25): GATTATAAAGACCATGATGGTGATTATAAGGATCATGATATCGATTATAAGGATGACGACGATAAA DNA v.10 (SEQ ID NO: 26): GATTATAAAGATCACGATGGCGATTATAAAGACCACGATATTGATTATAAAGACGACGATGACAAA DNA v.11 (SEQ ID NO: 27): GACTATAAAGACCACGATGGTGATTATAAAGATCACGACATCGACTACAAAGACGATGATGATAAA DNA v.12 (SEQ ID NO: 28): GACTACAAAGATCACGACGGCGATTATAAAGATCACGATATTGACTATAAAGATGACGATGATAAA DNA v.13 (SEQ ID NO: 29): GATTATAAAGACCATGATGGAGATTACAAAGATCATGATATTGACTATAAAGACGACGACGATAAA DNA v.14 (SEQ ID NO: 30): GATTATAAAGATCACGATGGTGACTACAAAGATCACGATATCGATTATAAAGACGATGACGATAAA DNA v .15 (SEQ ID NO: 31): GACTACAAAGATCACGATGGTGATTATAAAGACCATGATATTGATTACAAAGATGATGATGACAAA Protein (SEQ ID NO: 32): DYKDHDGDYKDHDIDYKDDDDK Peptide Linker Peptide v.1 (SEQ ID NO: 33): (GAS)_n Peptide linker v.2 (SEQ ID NO: 34): (GSA)_n Peptide linker v.3 (SEQ ID NO: 35): (G)_n ;n = 4-8 peptide linker v.4 (SEQ ID NO: 36): (GGGGS)_n ;n = 1-3 peptide linker v.5 (SEQ ID NO:37): VGKGGSGG peptide linker v.6 (SEQ ID NO:38): (PAPAP)_n Peptide linker v.7 (SEQ ID NO: 39): (EAAAK)_n ;n=1-3 peptide linker v.8 (SEQ ID NO:40): (AYL)_n Peptide linker v.9 (SEQ ID NO: 41): (LRA)_n Peptide linker v.10 (SEQ ID NO: 42): (RLRA)_n PEST-like sequence PEST-like sequence v.1 (SEQ ID NO: 43): KENSISSMAPPASPPASPKTPIEKKHADEIDK PEST-like sequence v.2 (SEQ ID NO: 44): KENSISSMAPPASPPASPK PEST-like sequence v.3 (SEQ ID NO: 45): KTEEQPSEVNTGPR PEST-like Sequence v.4 (SEQ ID NO:46): KESVVDASESDLDSSMQSADESTPQPLK PEST-like sequence v.5 (SEQ ID NO:47): KSEEVNASDFPPPPTDEELR PEST-like sequence v.6 (SEQ ID NO:48): RGGRPTSEEFSSLNSGDFTDDENSETTEEEIDR PEST-like sequence v.7 ( SEQ ID NO:49): KQNTASTETTTTNEQPK PEST-like sequence v.8 (SEQ ID NO:50): KQNTANTETTTTNEQPK PEST-like sequence v.9 (SEQ ID NO:51): RSEVTISPAETPESPPATP PEST-like sequence v.10 (SEQ ID NO:52 ): KASVTDTSEGDLDSSMQSADESTPQPLK PEST-like sequence v.11 (SEQ ID NO: 53): KNEEVNASDFPPPPTDEELR PEST-like sequence v.12 (SEQ ID NO: 54): RGGIPTSEEFSSLNSGDFTDDENSETTEEEIDR LLO protein LLO protein v.1 (SEQ ID NO: 55):LLO protein v.2 (SEQ ID NO: 56):N-terminal truncated LLO protein v, 1 (SEQ ID NO: 57):N-terminal truncated LLO protein v.2 (SEQ ID NO: 58):N-terminal truncated LLO protein v.3 (SEQ ID NO: 59):Nucleic acid encoding the N-terminal truncated LLO protein v.3 (SEQ ID NO: 60):ActA protein ActA protein v.1 (SEQ ID NO: 61)ActA protein v.2 (SEQ ID NO: 62)ActA fragment v.1 (SEQ ID NO: 63)ActA fragment v.2 (SEQ ID NO: 64)ActA fragment v.3 (SEQ ID NO: 65)ActA fragment v.4 (SEQ ID NO: 66)ActA fragment v.5 (SEQ ID NO: 67)Nucleic acid encoding ActA fragment v.5 (SEQ ID NO: 68)ActA fragment v.6 (SEQ ID NO: 69)ActA fragment v.7 (SEQ ID NO: 70)Nucleic acid encoding ActA fragment v.7 (SEQ ID NO: 71)ActA fragment fused to the Hly signal peptide (SEQ ID NO: 72)ActA substitution (SEQ ID NO: 73)LLO mutation LLO cholesterol binding domain (SEQ ID NO: 74)HLA-A2 Restriction epitope from NY-ESO-1 (SEQ ID NO: 75) Dal andDat Lm Alanine racemase (SEQ ID NO: 76) Lm D-amino acid transaminase (SEQ ID NO: 77)codingLm Nucleic acid of alanine racemase (SEQ ID NO: 78)codingLm D-amino acid transaminase nucleic acid (SEQ ID NO: 79) PrfA Wild type PrfA (SEQ ID NO: 80)Nucleic acid encoding wild-type PrfA (SEQ ID NO: 81)D133V PrfA (SEQ ID NO: 82)Nucleic acid encoding D133V PrfA (SEQ ID NO: 83)4×glycine linkage DNA sequence G1 (SEQ ID NO: 84)G2 (SEQ ID NO: 85)G3 (SEQ ID NO: 86)G4 (SEQ ID NO: 87)G5 (SEQ ID NO: 88)G6 (SEQ ID NO: 89)G7 (SEQ ID NO: 90)G8 (SEQ ID NO: 91)G9 (SEQ ID NO: 92)G10 (SEQ ID NO: 93)G11 (SEQ ID NO: 94)Dual HPV insertion sequence nucleic acid encoding 16 E7 (SEQ ID NO: 95)16 E7 (SEQ ID NO: 96)Nucleic acid encoding 18 E7 (SEQ ID NO: 97)18 E7 (SEQ ID NO: 98)Nucleic acid encoding the HPV16 E7-HPV18E7 insert (SEQ ID NO: 99)HPV16 E7-HPV18E7 insert (SEQ ID NO: 100)Nucleic acid encoding 16 E7-4×Gly-18 E7 (SEQ ID NO: 101)16 E7-4×Gly-18 E7 (SEQ ID NO: 102)Nucleic acid encoding 16 E7-18 E7-3×FLAG (SEQ ID NO: 103)16 E7-18 E7-3×FLAG (SEQ ID NO: 104)Nucleic acid encoding 16 E7-4×Gly-18 E7-3×FLAG (SEQ ID NO: 105)16 E7-4×Gly-18 E7-3×FLAG (SEQ ID NO: 106)Nucleic acid encoding 16 E7-18 E7-SIINFEKL (SEQ ID NO: 107)16 E7-18 E7-SIINFEKL (SEQ ID NO: 108)Nucleic acid encoding 16 E7-4×Gly-18 E7-SIINFEKL (SEQ ID NO: 109)16 E7-4×Gly-18 E7-SIINFEKL (SEQ ID NO: 110)Nucleic acid encoding 16 E7-18 E7-3×FLAG-SIINFEKL (SEQ ID NO: 111)16 E7-18 E7-3×FLAG-SIINFEKL (SEQ ID NO: 112)Nucleic acid encoding 16 E7-4×Gly-18 E7-3×FLAG-SIINFEKL (SEQ ID NO: 113)16 E7-4×Gly-18 E7-3×FLAG-SIINFEKL (SEQ ID NO: 114)dtLLO helper sequence detoxification of Listeria lysin O (dtLLO) (SEQ ID NO: 115)EXAMPLES Example 1. Relationship between 12-month total survival and genotype in AXAL Phase 2 study GOG-0265 [0220] GOG-0265 is a single-group, open-label, phase 2 multicenter study (NCT01266460) ), which was designed to evaluate axalimogene filolisbac (AXAL) in patients with persistent or recurrent metastatic (squamous or non-squamous cell) cervical cancer (PRmCC) in a standard Simon two-stage design Safety and activity in patients. AXAL is a live attenuated Listeria monocytogenes (Lm ), which is bioengineered to secrete HPV16 E7 protein fused to a truncated fragment of Listeria lysin O (tLLO). AXAL targets HPV-transformed cells to induce anti-tumor T cell immunity and disrupt immune tolerance in the tumor microenvironment. The first phase of the study included six patients with a run-in and 26 patients enrolled. In the second phase, 24 patients were enrolled. [0221] HPV genotyping was performed on samples of 36 of the 50 patients enrolled in GOG-0265. Among the 4 patients, the DNA was too poor to be genotyped, and in 1 patient, the amount of DNA was insufficient. Nine of the patients failed to obtain DNA. HPV was not detected in 4 of the patients. Of the remaining 32 patients, 14 were positive for HPV in the α9 family (12 patients were positive for HPV16; 2 patients were positive for HPV33), and 16 patients were positive for HPV in the α7 family (12 patients with HPV18) Positive; 4 patients were positive for HPV45), and 2 patients were positive for HPV of the α7 family and HPV of the α9 family (1 patient was positive for both HPV16 and HPV18, and 1 patient had both HPV16 and HPV45 Positive). [0222]Overall, the 12-month survival rate was 38% (19/50) among all 50 patients, and the 12-month survival rate was positive for patients with positive HPV (HPV16 or HPV33) tests of the α9 family. 57% (8 of 14 patients), the 12-month survival rate of patients who tested positive for HPV (HPV18 or HPV45) in the α7 family was 38% (6 of 16 patients) and was confirmed as HPV Positive (i.e., removal of HPV-negative patients or failure to obtain DNA samples or patients with poor quality or insufficient DNA samples) was 44% (15 of 36 patients). [0224] Based on the protocol of the patients enrolled in the study (n=50), the prognostic factor was defined and the expected 12-month survival rate was 25%. Treating this 25% 12-month overall survival rate with a 38% overall 12-month overall survival rate actually observed in the total study population, treatment with AXAL caused a 52% increase in the expected 12-month overall survival. [0225] AXAL is bioengineered to secrete E7 protein from HPV16. The 12-month overall survival rate for HPV16-positive patients was 57%, which was significantly higher than the expected 25% overall survival, consistent with AXAL induction of anti-tumor immune responses against HPV16-transformed cells. Unexpectedly, however, despite the fact that the HPV16 E7 protein is only 42% identical to the HPV18 E7 protein, it is not tested positive for HPV16 and HPV18 or HPV45 (not HPV16 or even HPV in the same HPV family as HPV16) The 12-month overall survival rate of patients who tested positive was 38%, which was 52% higher than the expected 12-month overall survival rate of 25%. Example 2. Construction of Lm-LLO-HPV16 E7-HPV18 E7 Since HPV16 and HPV18 are rarely found in the same patient, it has not previously been intended to place both HPV16 E7 and HPV18 E7 proteins together in a single immunotherapeutic vector. . However, in view of the unexpected results shown in Example 1, Listeria strains expressing both HPV16 E7 and HPV18 E7 will be produced by SEQ ID NOs: 99, 101, 103, 105, Any of 107, 109, 111, and 113 ligated into pGG55 or pGG55-based plastids downstream of the gene encoding the tLLO protein and fused to the gene, the pGG55 or pGG55-based plastid expressed by the hly promoter drive. The inserts set forth in SEQ ID NOS: 99, 101, 103, 105, 107, 109, 111 and 113 and their correspondingly encoded peptides are provided in Table 2. [0227]The resulting plasmid will be electroporated into a suitable Listeria strain. A suitable Listeria strain is lackingLm The transcriptional activator PrfA strain XFL-7, XFL-7 based Listeria strain or lackprfA It is similar to the Listeria strain. The smaller size of HPV18 E7 protein (105 amino acids; SEQ ID NO: 96) and the smaller size of HPV16 E7 protein (97 amino acids; SEQ ID NO: 98) make it possible to generate live attenuated Listeria monocytogenesLm ), which is bioengineered to secrete tLLO protein fused to HPV16 E7 and HPV18 E7 in tandem, even when limited by platform size. [0229] Other suitable Listeria strains are described, for example, in WO-2009/143167, WO-2016/011353, WO-2016/011320, WO-2010/102140, WO-2011/060260, WO-2013/025925, WO-2015/130810, WO-2015/167748, WO-2012/138377, WO-2012/125551, WO-2016/126876, WO-2013/138337, WO-1996-014087, WO-2006-036550, WO- 2008/140812, WO-1999/025376, WO-2001/072329, WO-2007/106476, WO-2007/130455, WO-2008/109155, WO-2010/008782, WO-2004-062597, WO-2015/ 164121, WO-2015/126921, WO-2015/134722, WO-2016/061182, WO-2016/011362, WO-2016/100924, WO-2016/011357, WO-2016/061277, WO-2016/100929, WO-2016/141121, WO-2016/126878, WO-2016/183361, WO-2016/191545, WO-2006/017856, WO-2008/130551, US-2011/0129499, US-2012/0135033, US- 2014/0234370, US-2014/0335120, US-2015/0098964, US-2015/0366955, US-2016/0361401, US-6,051,237, US-6,099,848, US-6,767,542, US-6,855,320, US-7,635,479, US- 7,662,396, US-7,794,729, US-7,820,180, US-8,114,414, US-8,337,861, US-8,771,702, US-8,778,329, U S-8, 791, 237, US-9, 012, 141, US-9, 017, 660, US-9, 017, 660, US-9, 226, 958, US-9, 463, 227, and PCT Application Nos. PCT/US2016/051748, PCT/US2016/052322, and PCT/US2016/057220, This is incorporated herein by reference. Example 3. In vivo experiment of ADXS-DUAL (ADXS-602) [0230] The murine HPV+ TC-1 tumor model was used to test the ability of ADXS-DUAL (ADXS-602) to control tumor growth and prolong the survival of animals. ADXS-DUAL (ADXS-602) represents a fusion protein containing E7 proteins from both HPV-16 and HPV-18. TC-1 tumor cells are derived from the C57BL/6 lung epithelial cell line, which is immortalized by HPV 16 E6 and E7 and transformed by activated ras oncogene. To establish a primary tumor, 1 × 105 TC-1 cells were subcutaneously injected into the flank of the flank of C57BL/6 mice, and allowed to grow for 5 days, after which treatment was started. Tumor-bearing mice received 1 × 108 CFU ADXS-602, 1 x 108 CFU XFL7 (a parent strain of ADXS-602 lacking tumor-associated antigen) or PBS at weekly intervals for a total of 3 doses (see figure 1). Tumor volume [(length × width × width) / 2] was measured twice a week. Kill mice with a tumor volume close to 2000 mm3. ADXS-DUAL exhibited significant tumor control (Figure 2) and survival (Figure 3) responses when compared to PBS and XFL-7 (empty Lm) vectors.

[0010] The following drawings form a part of this specification and are included to further illustrate the specific aspects of the invention. One or more can be better understood. This patent or application file contains at least one drawing of a color drawing. Upon request and payment of the necessary fee, the Patent Office will provide a copy of the patent or patent application publication with a color schema. [0011] FIG. 1 shows the study design of Example 3. [0012] Figure 2 shows the results of tumor volume. Mice treated with ADXS-DUAL (ADXS-602) showed a significant reduction in tumor volume. [0013] Figure 3 shows the results of percent survival. Mice treated with ADXS-DUAL (ADXS-602) showed a significant increase in the percentage of survival. [0014] It will be appreciated that the elements shown in the drawings are not necessarily to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. In addition, reference numbers may be repeated among the figures to indicate corresponding or similar elements.

Claims (61)

A recombinant Listeria (Listeria) strain which comprises a nucleic acid encoding a fusion polypeptide comprising a first open reading frame, wherein the fusion polypeptide comprises a PEST-containing peptide of an antigen peptide fused with HPV16 and HPV18 antigenic peptide, wherein the HPV16 The antigenic peptide and the HPV 18 antigen peptide are operably linked in series. The recombinant Listeria strain of claim 1, wherein the HPV16 antigen peptide is an HPV16 E6 antigen peptide or an HPV16 E7 antigen peptide, and wherein the HPV18 antigen peptide is an HPV18 E6 antigen peptide or an HPV18 E7 antigen peptide. The recombinant Listeria strain of claim 1, wherein the HPV 16 antigen peptide and the HPV 18 antigen peptide are directly fused to each other without an intervention sequence. The recombinant Listeria strain of claim 1, wherein the HPV16 antigen peptide and the HPV18 antigen peptide are linked to each other via a peptide linker. The recombinant Listeria strain of claim 4, wherein the peptide linkers comprise one or more of the linkers set forth in SEQ ID NOs: 33-42. The recombinant Listeria strain according to any one of claims 1 to 5, wherein the HPV16 antigen peptide is an HPV16 E7 antigen peptide, and the HPV18 antigen peptide is an HPV18 E7 antigen peptide. The recombinant Listeria strain of claim 6, wherein the HPV16 E7 antigen peptide comprises a sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:96 And/or wherein the HPV18 E7 antigen peptide comprises a sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:98. The recombinant Listeria strain of claim 7, wherein the segment encoding the open reading frame of the HPV16 E7 antigen peptide comprises at least 90%, 95%, 96%, 97%, 98% of SEQ ID NO: 95, a 99% or 100% consensus sequence and encoding the peptide sequence set forth in SEQ ID NO: 96, and/or wherein the open reading frame encoding the HPV18 E7 antigen peptide comprises at least 90% of SEQ ID NO: 97 a sequence of 95%, 96%, 97%, 98%, 99% or 100% identical and encoding the peptide sequence set forth in SEQ ID NO:98. The recombinant Listeria strain according to any one of claims 6 to 8, wherein the segment of the fusion polypeptide comprising the HPV 16 antigen peptide and the HPV 18 antigen peptide comprises at least 90% of the following sequence, Sequences of 95%, 96%, 97%, 98%, 99% or 100% identity: SEQ ID NOs: 100, 102, 104, 106, 108, 110, 112 and 114. The recombinant Listeria strain of claim 9, wherein the segment of the open reading frame encoding the segment of the fusion polypeptide comprising the HPV 16 antigen peptide and the HPV 18 antigen peptide comprises at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical sequence: SEQ ID NO: 99, 101, 103, 105, 107, 109, 111 or 113, and encodes the sequence set forth below: SEQ ID NO: 100, 102, 104, 106, 108, 110, 112 or 114. The recombinant Listeria strain according to any one of claims 1 to 5, wherein the HPV16 antigen peptide is an HPV16 E6 antigen peptide, and the HPV18 antigen peptide is an HPV18 E6 antigen peptide. The recombinant Listeria strain according to any one of claims 1 to 5, wherein the HPV16 antigen peptide is an HPV16 E6 antigen peptide, and the HPV18 antigen peptide is an HPV18 E7 antigen peptide. The recombinant Listeria strain according to any one of claims 1 to 5, wherein the HPV16 antigen peptide is an HPV16 E7 antigen peptide, and the HPV18 antigen peptide is an HPV18 E6 antigen peptide. The recombinant Listeria strain according to any one of the preceding claims, wherein the fusion polypeptide further comprises one of the N-terminal and/or C-terminal of the HPV 16 antigen peptide and the HPV 18 antigen peptide operably linked in series Or multiple peptide tags. The recombinant Listeria strain of claim 14, wherein the one or more peptide tags comprise one or more of the following: a 3 x FLAG tag; a 2 x FLAG tag, a 6 x His tag; and a SIINFEKL tag. The recombinant Listeria strain of claim 15, wherein the fusion polypeptide comprises a 3×FLAG tag at the N-terminus of the HPV16 antigen peptide operably linked in series and the HPV18 antigen peptide, and at the antigenic peptide SIINFEKL label on the C side. The recombinant Listeria strain of claim 15, wherein the fusion polypeptide comprises the SIINFEKL tag at the N-terminus of the HPV16 antigen peptide operably linked in series and the C-terminus of the antigen peptide 3 × FLAG tag. The recombinant Listeria strain of claim 15, wherein the fusion polypeptide comprises a 3xFLAG tag and a SIINFEKL tag at the C-terminus of the HPV16 antigen peptide operably linked in series and the HPV18 antigen peptide. The recombinant Listeria strain of any one of the preceding claims, wherein the PEST-containing peptide is at the N-terminus of the fusion polypeptide. The recombinant Listeria strain according to any one of the preceding claims, wherein the PEST-containing peptide is a Listeria lysin O (LLO, listeriolysin O) protein or a fragment thereof or an ActA protein or a fragment thereof. The recombinant Listeria strain of claim 20, wherein the Listeria lysin O (LLO) protein or a fragment thereof is an N-terminal fragment of LLO. The recombinant Listeria strain of claim 21, wherein the N-terminal fragment of the LLO has the sequence set forth in any one of SEQ ID NOs: 57-59. The recombinant Listeria strain of claim 20, wherein the PEST-containing peptide is the LLO protein or a fragment thereof and comprises a mutation in a cholesterol binding domain. The recombinant Listeria strain of claim 23, wherein the LLO mutation comprises one of: (1) a substitution of residue C484, W491 or W492 of SEQ ID NO: 55 or when the LLO protein and SEQ ID NO: 55 is the corresponding substitution at the optimal alignment; or (2) the deletion of 1-11 amino acids in residues 483-493 of SEQ ID NO: 55 or when the LLO protein and SEQ ID NO: 55 The corresponding alignment is missing. A recombinant Listeria strain according to any one of the preceding claims, wherein the nucleic acid is operably integrated into the Listeria genome. The recombinant Listeria strain of any one of claims 1 to 24, wherein the nucleic acid is in an episomal form. A recombinant Listeria strain according to any one of the preceding claims, wherein the nucleic acid does not confer antibiotic resistance to the recombinant Listeria strain. The recombinant Listeria strain of any one of the preceding claims, wherein the recombinant Listeria strain is attenuated. The recombinant Listeria strain according to any one of the preceding claims, wherein the recombinant Listeria strain is an auxotrophic Listeria strain. The recombinant Listeria strain of claim 28 or 29, wherein the attenuated Listeria strain comprises a mutation in one or more endogenous genes that inactivates the one or more endogenous genes. The recombinant Listeria strain of claim 30, wherein the one or more endogenous genes comprise prfA . The recombinant Listeria strain of claim 30, wherein the one or more endogenous genes comprise actA . The recombinant Listeria strain of claim 30, wherein the one or more endogenous genes comprise actA and inlB . The recombinant Listeria strain of claim 30, wherein the one or more endogenous genes comprise actA , dal , and dat . A recombinant Listeria strain according to any one of the preceding claims, wherein the nucleic acid comprises a second open reading frame encoding a metabolic enzyme. A recombinant Listeria strain according to claim 35, wherein the metabolic enzyme is a leucine racemase or a D-amino acid transaminase. A recombinant Listeria strain according to any one of the preceding claims, wherein the fusion polypeptide is expressed from a hly promoter, a prfA promoter, an actA promoter or a p60 promoter. The recombinant Listeria strain of claim 37, wherein the fusion polypeptide is expressed from a hly promoter. The recombinant Listeria strain according to any one of the preceding claims, wherein the recombinant Listeria strain is a recombinant Listeria monocytogenes strain. The recombinant Listeria strain according to any one of claims 1 to 19, wherein the recombinant Listeria strain is an attenuated mononuclear granulating Listeria comprising a deletion of prfA or an inactivating mutation in prfA A bacterial strain, wherein the nucleic acid is in an episomal plastid and comprises a second open reading frame encoding a D133V PrfA mutant protein. The requested item 1 to 19 of a recombinant Listeria strain, wherein the strain is a recombinant Listeria comprising the actA, or deletion of the actA dal and the dat, dal and the dat inactivating mutations in attenuated a Listeria monocytogenes strain, wherein the nucleic acid is in an episomal plastid and comprises a second open reading frame encoding an alanine racemase or a D-amino acid transaminase, and wherein the The peptide of PEST is the N-terminal fragment of LLO. The recombinant Listeria strain of any one of claims 1 to 19, wherein the recombinant Listeria strain is an attenuated strain of Listeria monocytogenes comprising a deletion or inactivating mutation of actA and inlB Wherein the nucleic acid is integrated by genome, and wherein the PEST-containing peptide is an ActA protein or a fragment thereof. The recombinant Listeria strain of any one of the preceding claims, wherein the recombinant Listeria strain has been subcultured by an animal host. A recombinant Listeria strain according to any one of the preceding claims, wherein the recombinant Listeria strain is capable of escaping phagosomes. An immunogenic composition comprising the recombinant Listeria strain of any of the preceding claims. The immunogenic composition of claim 45, wherein the immunogenic composition further comprises an adjuvant. The immunogenic composition of claim 46, wherein the adjuvant comprises a granule ball/macrophage colony stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphorus Mercapto lipid A, dtLLO or an oligonucleotide containing unmethylated CpG. A method of inducing an immune response against a tumor or cancer in an individual, comprising administering to the individual a recombinant Listeria strain of any one of claims 1 to 44 or immunizing any one of claims 45 to 47 Original composition. A method of preventing or treating a tumor or cancer in an individual, comprising administering to the individual the immunogenicity of the recombinant Listeria strain of any one of claims 1 to 44 or any one of claims 45 to 47 Composition. The method of any one of claims 48 to 49, wherein the method further comprises administering an immunological checkpoint inhibitor antagonist. The method of claim 50, wherein the immunological checkpoint inhibitor comprises an anti-PD-1 antibody or antigen-binding fragment thereof and/or an anti-CTLA-4 antibody or antigen-binding fragment thereof. The method of any one of clauses 48 to 51, wherein the method further comprises administering a T cell stimulating agent. The method of claim 52, wherein the T cell stimulating agent comprises an anti-OX40 antibody or antigen-binding fragment thereof or an anti-GITR antibody or antigen-binding fragment thereof. The method of any one of claims 48 to 53, wherein the tumor or cancer is a cervical tumor or cancer, an anal tumor or cancer, a head and neck tumor or a cancer or an oropharyngeal tumor or cancer. The method of claim 54, wherein the tumor or cancer is a cancer metastasis. The method of any one of claims 48 to 55, wherein the tumor or cancer is HPV16 positive. The method of any one of claims 48 to 55, wherein the tumor or cancer is HPV18 positive. A cell bank comprising one or more recombinant Listeria strains of any one of claims 1 to 44. The cell bank of claim 58, wherein the cell bank is a frozen cell bank or a lyophilized cell bank. The recombinant Listeria strain of any one of claims 6 to 8, wherein the segment of the fusion polypeptide comprising the HPV 16 antigen peptide and the HPV 18 antigen peptide comprises SEQ ID NO: 102. The recombinant Listeria strain of claim 9, wherein the open reading frame segment encoding the segment of the fusion polypeptide comprising the HPV 16 antigen peptide and the HPV 18 antigen peptide comprises SEQ ID NO: 101 and is encoded accordingly The sequence set forth in SEQ ID NO:102.